Kinase suppressor of Ras inactivation for therapy of Ras mediated tumorigenesis

ABSTRACT

The present invention relates to methods and compositions for the specific inhibition of kinase suppressor of Ras (KSR). In particular, the invention provides genetic approaches and nucleic acids for the specific inhibition of KSR, particularly of KSR expression. The invention relates to antisense oligonucleotides and the expression of nucleic acid which is substantially complementary to KSR RNA. Oligonucleotide and nucleic acid compositions aree provided. The invention provides methods to inhibit KSR, including inhibition of KSR expression. Methods for blocking gƒ Ras mediated tumorigenesis, metastasis, and for cancer therapy are provided. Methods for conferring radiosensitivity to cells are also provided.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a Continuation-in-Part of co-pending PCTApplication NO. PCT/US03/16961 filed May 29, 2003, which in turn, claimspriority from U.S. Provisional Application Ser. No. 60/384,228, filedMay 30, 2002, and U.S. Provisional Application Ser. No. 60/460,023,filed Apr. 3, 2003. Applicants claim the benefits of 35 U.S.C. § 120 asto the PCT application and priority under U.S.C. § 119(e) as to saidUnited States Provisional applications, and the entire disclosures ofall of which applications are incorporated herein by reference in theirentireties.

GOVERNMENTAL SUPPORT

The research leading to the present invention was supported, at least inpart, by a grant from the National Institutes of Health, GrantNo.CA42385 and Grant No.CA52462. Accordingly, the Government may havecertain rights in the invention.

FIELD OF THE INVENTION

The present invention relates to methods and compositions for thespecific inhibition of of kinase suppressor of Ras (KSR). In particular,the invention provides genetic approaches and nucleic acids for thespecific inhibition of KSR, particularly of KSR expression. Theinvention relates to antisense oligonucleotides and the expression ofnucleic acid complementary to KSR RNA to specifically inhibit KSR andblock gƒRas mediated tumorigenesis.

BACKGROUND OF THE INVENTION

Ras plays an essential role in oncogenic transformation and genesis.Oncogenic H-, K-, and N-Ras arise from point mutations limited to asmall number of sites (amino acids 12, 13, 59 and 61). Unlike normalRas, oncogenic ras proteins lack intrinsic GTPase activity and henceremain constitutively activated (Trahey, M., and McCormick, F. (1987)Science 238: 542-5; Tabin, C. J. et al. (1982) Nature. 300: 143-9;Taparowsky, E. et al. (1982) Nature. 300: 762-5). The participation ofoncogenic ras in human cancers is estimated to be 30% (Almoguera, C. etal (1988) Cell. 53:549-54).

Mutations are frequently limited to only one of the ras genes, and thefrequency is tissue- and tumor type-specific. K-ras is the most commonlymutated oncogene in human cancers, especially the codon-12 mutation.While oncogenic activation of H-, K-, and N-Ras arising from singlenucleotide substitutions has been observed in 30% of human cancers (Bos,J. L. (1989) Cancer Res 49, 4682-9), over 90% of human pancreatic cancermanifest the codon 12 K-ras mutation (Almoguera, C. et al. (1988) Cell53, 549-54; Smit, V. T. et al. (1988) Nucleic Acids Res 16, 7773-82;Bos, J. L. (1989) Cancer Res 49, 4682-9). Pancreatic ductaladenocarcinoma, the most common cancer of the pancreas, is notorious forits rapid onset and resistance to treatment. The high frequency of K-rasmutations in human pancreatic tumors suggests that constitutive Rasactivation plays a critical role during pancreatic oncogenesis.Adenocarcinoma of the exocrine pancreas represents the fourth-leadingcause of cancer-related mortality in Western countries. Treatment hashad limited success and the five-year survival remains less than 5% witha mean survival of 4 months for patients with surgically unresectabletumors (Jemal, A et al (2002) CA Cancer J Clin 52, 23-47; Burris, H. A.,3rd et al. (1997) J Clin Oncol 15, 2403-13). This point mutation can beidentified early in the course of the disease when normal cuboidalpancreatic ductal epithelium progresses to a flat hyperplastic lesion,and is considered causative in the pathogenesis of pancreatic cancer(Hruban, R. H. et al (2000) Clin Cancer Res 6, 2969-72; Tada, M. et al.(1996) Gastroenterology 110, 227-31). The regulation of oncogenic K-rassignaling in human pancreatic cancer, however, remains largely unknown.

K-ras mutations are present in 50% of the cancers of colon and lung(Bos, J. L. et al. (1987) Nature. 327: 293-7; Rodenhuis, S. et al.(1988) Cancer Res. 48: 5738-41). In cancers of the urinary tract andbladder, mutations are primarily in the H-ras gene (Fujita, J. et al.(1984) Nature. 309: 464-6; Visvanathan, K. V. et al. (1988) OncogeneRes. 3: 77-86). N-ras gene mutations are present in 30% of leukemia andliver cancer. Approximately 25% of skin lesions in humans involvemutations of the Ha-Ras (25% for squamous cell carcinoma and 28% formelanomas) (Bos, J. L. (1989) Cancer Res. 49:4683-9; Migley, R. S. andKerr, D. J. (2002) Crit Rev Oncol Hematol. 44:109-20).50-60% of thyroidcarcinomas are unique in having mutations in all three genes (Adjei, A.A. (2001) J Natl Cancer Inst. 93: 1062-74).

Constitutive activation of Ras can be achieved through oncogenicmutations or via hyperactivated growth factor receptors such as theEGFRs. Elevated expression and/or amplification of the members of theEGFR family, especially the EGFR and HER2, have been implicated invarious forms of human malignancies (as reviewed in Prenzel, N. et al.(2001) Endocr Relat Cancer. 8: 11-31). In some of these cancers(including pancreas, colon, bladder, lung), EGFR/HER2 overexpression iscompounded by the presence of oncogenic Ras mutations. Abnormalactivation of these receptors in tumors can be attributed tooverexpression, gene amplification, constitutive activation mutations orautocrine growth factor loops (Voldborg, B. R. et al. (1997) Ann Oncol.8:1197-206). For growth factor receptors, especially the EGFRs,amplification or/and overexpression of these receptors frequently occurin the cancers of the breast, ovary, stomach, esophagus, pancreatic,lung, colon neuroblastoma.

While various therapeutic strategies have been developed to inactivatekey components of the Ras-Raf-MAPK cascade, specific inhibition ofgain-of-function or constitutive Ras (gƒRas) action has not beenachieved clinically (Adjei, A. A. (2001) J Natl Cancer Inst 93, 1062-74;Cox, A. D. & Der, C. J. (2002) Curr Opin Pharmacol 2, 388-93).

Therefore, in view of the aforementioned deficiencies attendant withprior art methods to inactivate or inhibit the Ras pathway, andparticularly Ras-mediated cancers, it should be apparent that therestill exists a need in the art for methods and compositions for specificinhibition of the Ras pathway and particularly for inhibition of gƒRas.

The citation of references herein shall not be construed as an admissionthat such is prior art to the present invention.

SUMMARY OF THE INVENTION

The present invention relates to methods and compositions for thespecific inhibition of kinase suppressor of Ras (KSR). The compositionsand methods of the present invention inhibit the expression and/oractivity of KSR. In particular, the invention provides geneticapproaches and nucleic acids for the specific inhibition of KSR. It isherein demonstrated that on specific inhibition of KSR the Ras pathwayis disrupted and, specifically, Ras-mediated tumors and tumorigenesis isinhibited or blocked, existing tumors regress, metasasis is inhibitedand proliferation of tumor or cancer cells is inhibited.

The present invention provides oligonucleotides and nucleic acids whichspecifically inhibit or block the expression and activity of KSR. Inparticular, antisense oligonucleotides and the expression of nucleicacid complementary to KSR RNA specifically inhibits expression of KSRand blocks gƒRas mediated tumorigenesis.

The present invention provides an oligonucleotide which is substantiallycomplementary to a region of KSR RNA, wherein said oligonucleotideinhibits the expression of KSR. The invention further provides anoligonucleotide which is substantially complementary to a nucleic acidencoding mammalian KSR. In particular embodiments, oligonucleotides areprovided which are substantially complementary to nucleic acid encodingmammalian KSR, particularly human and mouse KSR.

In an aspect of the invention an oligonucleotide is provided which issubstantially complementary to a translation initiation site, 5′untranslated region, coding region or 3′ untranslated region of mRNAencoding mammalian KSR. In one such aspect, the invention thus providesan oligonucleotide which is substantially complementary to a translationinitiation site, 5′ untranslated region, coding region or 3′untranslated region of mRNA encoding human KSR as provided in FIG. 14and SEQ ID NO: 24. In a particular embodiment, the invention provides anantisense oligonucleotide substantially complementary to the N-terminalcoding region of mammalian KSR mRNA, particularly to the region ofnucleotides 1 through 761 of the coding region of mammalian KSR mRNA. Inone embodiment, the invention includes an antisense oligonucleotidecomprising a sequence substantially complementary to the CA1 region ofKSR. The invention provides oligonucleotides comprising a sequencesubstantially complementary to nucleotides encoding amino acids 33 to 72of the sequence of human KSR and amino acids 42 to 82 of the sequence ofmouse KSR, or a portion thereof.

In a further embodiment, the invention includes an antisenseoligonucleotide complising a sequence substantially complementary tonucleotides 97 to 216 of the coding sequence of human KSR (SEQ ID NO:25) or nucleotides 124 to 243 of the coding sequence of mouse KSR (SEQID NO: 1), or a portion thereof, such nucleotides encoding amino acids33 to 72 of human KSR (SEQ ID NO: 26) and amino acids 42 to 82 of mouseKSR (SEQ ID NO:2) or a portion thereof. In particular, oligonucleotidesof the invention include oligonucleotides comprising a sequencesubstantially complementary to nucleotides selected from the group of:nucleotides 124 to 141 of human KSR, corresponding to nucleotides 151 to168 of mouse KSR (SEQ ID NO: 3); nucleotides 154 to 171 of human KSR(SEQ ID NO: 27), corresponding nearly identically (with a single basepair difference at the 5′ most nucleotide) to nucleotides 181 to 198 ofmouse KSR (SEQ ID NO:4); and nucleotides 187 to 204 of human KSR,corresponding to nucleotides 214 to 231 of mouse KSR (SEQ ID NO:5). Theinvention includes antisense oligonucleotides comprising a sequenceselected from the group of SEQ ID NOS: 6-8 and SEQ ID NOS: 29-38.

The oligonucleotides of the present invention may be labeled with adetectable label. In particular aspects, the label may be selected fromenzymes, ligands, chemicals which fluoresce and radioactive elements. Inthe instance where a radioactive label, such as the isotopes ³H, ¹⁴C,³²P, ³⁵S, ³⁶Cl, ⁵¹Cr, ⁵⁷Co, ⁵⁸Co, ⁵⁹Fe, ⁹⁰Y, ¹²⁵I, ¹³¹I, and ¹⁸⁶Re areused, known currently available counting procedures may be utilized. Inthe instance where the label is an enzyme, detection may be accomplishedby any of the presently utilized colorimetric, spectrophotometric,fluorospectrophotometric, amperometric or gasometric techniques known inthe art.

In a particular aspect, the nucleic acids and oligonucleotides of thepresent invention may be modified, either by manipulation of thechemical backbone of the nucleic acids or by covalent or non-covalentattachment of other moieties. In each or any case, such manipulation orattachment may serve to modify the stability, cellular, tissue or organuptake, or otherwise enhance efficacy of the nucleic acids andoligonucleotides. In further aspects of the invention, theoligonucleotides may be covalently linked to other molecules, includingbut not limited to polypeptides, carbohydrates, lipid or lipid-likemoieties, ligands, chemical agents or compounds, which may serve toenhance the uptake, stability or to target the oligonucleotides.

In further embodiments, the oligonucleotides of the present inventionare modified in their chemical backbone. In a particular embodiment, theoligonucleotides comprise at least one phosphorothioate (P-S) linkage.

Recombinant DNA molecules comprising a nucleic acid sequence whichencodes on transcription an antisense RNA complementary to mammalian KSRRNA or a portion thereof are provided by the invention. Further, therecombinant DNA molecules comprise a nucleic acid sequence wherein saidnucleic acid sequence is operatively linked to a transcription controlsequence. Cell lines transfected with these recombinant DNA moleculesare also included in the invention.

In a further aspect, an expression vector is provided which is capableof expressing a nucleic acid which is substantially complementary to thecoding sequence of KSR RNA, or a portion thereof, wherein said nucleicacid inhibits the expression of KSR. In a particular aspect, thisincludes an expression vector capable of expressing an oligonucleotidewhich is substantially complementary to the CA1 region of the codingsequence of KSR RNA, particularly of mouse or human KSR (SEQ ID NO: 1 orSEQ ID NO: 25), or a portion thereof, wherein said oligonucleotideinhibits the expression of KSR.

Compositions of the nucleic acids and oligonucleotides are an additionalaspect of the invention. The invention includes a composition comprisingan oligonucleotide which is substantially complementary to a region ofKSR RNA and a pharmaceutically acceptable carrier or diluent. Theinvention thus provides a pharmaceutical composition comprising atherapeutically effective amount of an oligonucleotide which issubstantially complementary to a region of KSR RNA and apharmaceutically acceptable carrier or diluent.

In a further aspect, compositions are provided comprising one or morechemotherapeutic or radiotherapeutic agent and an oligonucleotide whichis targeted to a mRNA encoding mammalian KSR and which inhibits KSRexpression.

In an additional embodiment, the invention provides a compositioncomprising an expression vector and a pharmaceutically acceptablecarrier or diluent, wherein said expression vector is capable ofexpressing nucleic acid which is substantially complementary to thecoding sequence of KSR RNA, or a portion thereof, wherein said nucleicacid inhibits the expression of KSR.

Methods for inhibiting expression of KSR are provided. In one aspect, amethod of inhibiting the expression of mammalian KSR comprisingcontacting cells which express KSR with an effective amount of a nucleicacid which is complementary to a portion of the mRNA encoding KSR isincluded. In particular, a method of inhibiting the expression ofmammalian KSR is provided, comprising contacting cells which express KSRwith an effective amount of the oligonucleotide of the present inventionwhereby expression of mammalian KSR is inhibited. In an additionalaspect, a method of inhibiting expression of KSR is provided, whereintissues or a tumor, particularly a tissue or tumor expressing gƒRas orwherein the Ras pathway is hyperactivated or Ras is overexpressed oramplified, is contacted with an effective amount of the oligonucleotideor nucleic acid of the present invention, thus inhibiting the expressionof KSR.

In a further embodiment, the invention provides compositions and methodsfor the inhibition or blockage of the activity of KSR, including thekinase or phosphorylation activity of KSR. In an additional aspect, amethod of inhibiting expression of KSR is provided, wherein a tumor,tissue or cells expressing KSR, particularly a tissue or tumorexpressing gƒRas or wherein the Ras pathway is hyperactivated or Ras isoverexpressed or amplified, is contacted with an effective amount of thenucleic acid or composition of the present invention, thus inhibitingthe activity of KSR.

The invention further includes a method of treating or preventing ahyperproliferative condition associated with the expression of gƒ-Ras orheightened expression of Ras in a mammal comprising administering tosaid mammal a therapeutically effective amount of a compound or agentwhich inhibits the expression of mammalian KSR protein. In one aspect ofthis method, said compound or agent is an antisense oligonucleotidewhich specifically hybridizes to a portion of the mRNA encoding KSR.

A method of treating or preventing a hyperproliferative conditionassociated with the expression of gƒ-Ras or heightened expression orhyperactivation of Ras in a mammal is provided, comprising expressing insaid mammal or administering to said mammal a therapeutically effectiveamount of a nucleic acid which is complementary to a portion of the mRNAencoding KSR.

In a further aspect, a method of treating or inhibiting the progressionof cancer in a mammal is included, comprising administering to a mammala therapeutically effective amount of a compound or agent which inhibitsthe expression of mammalian KSR protein. Cancers which are susceptibleto the invention's method include cancer selected from the group ofpancreatic cancer, lung cancer, skin cancer, urinary tract cancer,bladder cancer, liver cancer, thyroid cancer, colon cancer, intestinalcancer, leukemia, lymphoma, neuroblastoma, head and neck cancer, breastcancer, ovarian cancer, stomach cancer, esophageal cancer and prostatecancer.

Thus, a method is provided for treating or inhibiting the progression ofcancer in a mammal comprising administering to a mammal atherapeutically effective amount of one or more oligonucleotide of thepresent invention.

In addition, a method is provided for identifying compounds or agentswhich inhibit the expression of KSR comprising the steps of:

-   -   (a) incubating a cell expressing KSR in the presence and absence        of a candidate compound or agent; and    -   (b) detecting or measuring the expression of KSR in the presence        and absence of a candidate compound or agent,        whereby a decrease in the expression of KSR in the presence of        said candidate compound or agent versus in the absence of said        candidate compound or agent indicates that said compound or        agent inhibits the expression of KSR.

The invention includes additional compositions which can inhibit theexpression of a protein, in particular KSR, at the transcriptional levelby blocking translation of KSR mRNA or by facilitating destruction ordestabilization of the RNA such that translation cannot efficiently takeplace. In this aspect, the invention provides a ribozyme that cleavesKSR mRNA.

The present invention naturally contemplates several means forpreparation of the nucleic acids and oligonucleotides of the presentinvention, including as illustrated herein known recombinant techniques,and the invention is accordingly intended to cover such syntheticpreparations within its scope. The knowledge of the cDNA and amino acidsequences of KSR as disclosed herein facilitates the preparation of thenucleic acids of the invention by such recombinant techniques, andaccordingly, the invention extends to expression vectors prepared fromthe disclosed DNA sequences for expression in host systems byrecombinant DNA techniques, and to the resulting transformed hosts.

Other objects and advantages will become apparent to those skilled inthe art from a review of the following description which proceeds withreference to the following illustrative drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts targeted disruption of the ksr gene in mice. A, Strategyfor targeting the ksr allele. Simplified restriction maps of the 5′region of the wild-type ksr allele, the targeting vector, and themutated allele are shown. Homologous recombination with endogenous ksrreplaces an internal 1.1-kb SmaI-SpeI genomic fragment with a Neocassette. B, Southern blot analysis of an ES clone showing the correctinsertion of the targeting construct. Genomic DNA isolated from ES cellswas digested with BglII and XhoI and hybridized to the 5′ probe locatedjust outside the 5′ arm of the ksr targeting region as shown in A. Thewild-type allele yields a 5.7-kb fragment whereas the mutant alleleyields a 3.1-kb fragment. C, Genotyping of ksr^(−/−) mice by PCR. Thesize of the PCR product is 493 bp for the wt allele and 312 bp for themutated allele. D, Expression of ksr in wild type mouse embryos. Thesizes of the two transcripts are 6.4 kb and 7.4 kb. E, Northern blotanalysis of tissue ksr mRNAs. Poly-A⁺ RNA, isolated from differenttissues of adult ksr^(+/+), ksr^(+/−), and ksr^(−/−) mice, washybridized with a probe corresponding to domains CA2-CA4 in ksr cDNA.mRNA from NIH3T3 cells was used as control. F, KSR protein expression.Lysates prepared from wild-type and ksr^(−/−) tissues were analyzed bywestern blot with a specific anti-KSR monoclonal antibody. Note thatbrain expresses the slightly shorter B-KSR1 isoform while lung andspleen express the longer KSR1 isoform. Lysates were also prepared fromtwo independent sets of ksr^(+/+) and ksr^(−/−) MEFs. Equal loading wasconfirmed by reprobing blots with an anti-β-tubulin antibody.

FIG. 2 depicts skin phenotype in newbom ksr^(−/−) mice. Full thicknessskin cuts of 10-day old ksr^(+/+), ksr^(−/−) and egfr^(−/−) mice weresectioned 4-6 μm thick, placed on glass slides, and stained withhematoxylin and eosin. s-serpentine, bl-blister, do-disoriented.

FIG. 3 depicts defects in EGF- and TPA-induced MAPK signaling andproliferation in ksr^(−/−) MEFs. A, Western blot analysis of MAPKactivity upon EGF and TPA treatments. Low-passage MEFs derived fromksr^(+/+) and ksr^(−/−) were made quiescent by 48 h incubation inserum-free medium and stimulated with low doses of EGF for 3 min(upperpanel) or with TPA for 10 min (lower panel). Cells were lysed in NP40buffer and activation of the MAPK cascade was examined by western blotwith anti-phospho specific antibodies for the activated forms ofMAPK(ERK½). Shown are representative blots from one of four independentexperiments. B, Activation of endogenous Raf-1 upon EGF (upper panel)and TPA (lower panel) treatments were determined by Raf-1 activity assayas described herein in Methods. MEK1 phosphorylation was examined bywestern blot with anti-phospho specific antibodies for the activatedforms of MEK1. Shown are representative blots from one or fourindependent experiments. C, Proliferation of MEFs. 0.15×10⁶ ksr^(+/+) orksr^(−/−) low-passage MEFs were seeded on 60 mm plates and grown asdescribed in Methods. Cells were trypsinized every other day and countedby hemacytometer. Data (mean±SD) are compiled from three independentexperiments.

FIG. 4. Disruption of ksr gene abrogated oncogenic Ras-mediatedtumorigenesis in ksr^(−/−) mice. A, RT-PCR detection of ν-Ha-rasexpression from total RNA isolated from the epidermis of Tg.AC/ksr^(+/+)and Tg.AC/ksr^(−/−) mice following TPA treatment. Intron spanningprimers specific for the 3′UTR region of the ν-Ha-ras transgene wereused. The larger 279 bp amplicon, detected in the absence of reversetranscriptase [RT(−)], is derived from DNA and unspliced RNA. Thesmaller 214 bp amplicon is derived from spliced mRNA and is indicativeof transgene expression. B, Mice, grouped according to genotype(10/group), were treated with 5 μg of TPA twice a week for 15 weeks.Papillomas were counted weekly for 20 weeks.

FIG. 5. Inducible A431-Tet-Off-pTRE-KSR cells. A and B, Western blotanalysis of wild type Flag-KSR-S and Flag-DN-KSR expression (A), andinhibition of endogenous KSR1 expression by KSR-AS (B). Flag-KSR-S andDN-KSR were immunoprecipitated (IP) with the monoclonal anti-Flag (M2)antibody and detected by WB. The identity of Flag-KSR was confirmed byre-probing with a monoclonal anti-KSR antibody (BD Biosciences).Endogenous KSR1 was immunoprecipitated as described in Methods anddetected as above. C, Dose-dependent inhibition of Flag-KSR-S expressionby doxycycline. KSR-S cells were treated with indicated doses of Dox for24 h and Flag-KSR-S expression after Dox treatment was determined by WBas above. D and E, Inactivation of KSR1 by KSR-AS or DN-KSR leads toalterations in morphology (D), and the development of a multinucleiphenotype (E). A431-pTRE cells were examined under the phase-contrastmicroscope and photographed at 20× magnification for cell morphology (D)and 40× magnification for multinucleation (E).

FIG. 6. Inactivation of KSR1 abolishes EGF-stimulated biologicalresponses in A431 cells. A, Cell proliferation assay without (i) andwith (ii) EGF stimulation. Proliferation assays were performed asdescribed in Methods. B, Cell cycle distribution of A431-pTRE cells wasdetermined by FACS analysis as described in Methods. The proportion ofcells in the different phases of the cell cycle was calculated from theexperimental fluorescence histograms. C, Matrigel invasion assay inresponse to EGF stimulation. To optimize the stimulatory effect ofoverexpression of KSR-S on A431 cell invasion, the assay was terminatedafter 12 h (i). To maximize the inhibitory effect of KSR-AS and DN-KSRon A431 cell invasion, the assay was terminated after 18 h (ii). D, Softagar colony formation assays in response to EGF stimulation wereperformed as in Methods. For each cell line or treatment, 4 plates werecounted. These results represent one of four similar studies.

FIG. 7. Inactivation of KSR1 prevents A431 tumorigenesis. A, Growthcurve of A431 tumors. 10⁶ A431-pTRE cells were injected s.c. into nudemice as described in Methods. To determine the specificity of KSR-S onA431 tumorigenesis, Dox (100 mg/ml) was added to the drinking water of agroup of KSR-S tumor-bearing mice (KSR-S+Dox) 3 days prior to tumorimplantation and continued throughout the experiment to turn off KSR-Sexpression. Mice receiving KSR-AS and DN-KSR cells were monitored up to120 days. These results represent one of three similar experiments.There were 5 mice in each experimental group. B, H&E staining of A431tumors. Formalin-fixed, paraffin-embedded and 5 mm-cut A431-pTRE tumorsections were stained with H&E as described in Methods. Black arrows in(i) and (ii) indicate squamous differentiation. Black arrows in (iii)and (iv) indicate multinucleated tumor cells, and (v) is the enlargementof the framed field in (iii) of a multinucleated cell.

FIG. 8. Inactivation of KSR1 by AS-ODN attenuates A431 tumorigenesis. A,Immunofluorescence staining of endogenous KSR1 expression aftertreatment with 1 mM Control- or AS-ODNs was performed as in Methods.Nuclei were counter stained with DAPI. To compare the intensity offluorescence labeling, all images of KSR expression were taken with thesame exposure time. B and C, Dose-dependent inhibition of A431 cellproliferation (B) and invasion (C) by AS-ODN treatment. For theproliferation assay, 30% confluent A431 cells were treated with theindicated doses of Control- or AS-ODNs as in FIG. 3. Cell proliferationafter ODN treatment was calculated as percent of non-treated controls.Invasion assays were set up after 48 h of ODN treatment as above. D,Attenuation of A431 tumorigenesis by continuous infusion of AS-ODN at 5mg/kg/day. A431 seed tumor fragments freshly prepared as described inMethods, were transplanted s.c. into the right lateral flank of nudemice. Continuous infusion of ODNs was initiated 2 days prior to tumortransplantation. There were 5 mice in each treatment group. Theseresults represent one of three similar experiments.

FIG. 9. Inactivation of KSR1 by AS-ODN inhibits oncogenic K-rcissignaling in vitro in PANC-1. A, Dose-dependent inhibition of PANC-1cell proliferation by AS-ODN treatment. PANC-1 cells were treated withthe indicated doses of Control- or AS-ODNs and cell proliferation assayswere performed as in FIG. 7. B, AS-ODN treatment (5 μM) attenuated theproliferation of a panel of human pancreatic cancer cell lines. Theseeding density for each cell lines was determined in preliminarystudies so that all cell lines were 30-40% confluent when transfectedwith ODNs. C and D, c-Raf-1 is epistatic to KSR1. PANC-1 cells werefirst treated with Sense- or AS-ODNs for 48 h and then transfected withthe BXB-Raf as in Methods. 48 h after transfection, invasion and colonyformation assays were set up as in FIG. 7. The inhibitory effect ofAS-ODN on PANC-1 cell invasion (C) and transformation (D) was reversedby dominant positive BXB-Raf. E, AS-ODN treatment inhibited endogenousKSR1 expression in PANC-1 cells. Endogenous KSR1 was immunoprecipitatedfrom non-treated (NT), Sense-ODN-treated or AS-ODN-treated PANC-1 cells,and KSR1 expression was determined by WB as described in Methods.Purified Flag-KSR served as a positive control for the WB. F, MAPK andPI-3 kinase activation in AS-ODN-treated and BXB-Raf-1-transfectedPANC-1 cells in response to EGF were determined by WB analysis usingphospho-MAPK and phospho-Akt specific antibodies as described inMethods. Uuder these conditions, b-actin and total Akt were unchanged(not shown). These results represent one of three similar experiments.

FIG. 10. AS-ODN treatment abolished PANC-1 and A549 tumorigenesis invivo. A, Continuous infusion of AS-ODN abolished PANC-1 tumor growth.PANC-1 xenografts derived either from 10⁶ PANC-1 cells (i), or fromfreshly harvested seed PANC-1 tumors (ii) were transplanted into nudemice as described in Methods. A (i), established PANC tumors(approximately 100 mm³) were treated with 10 mg/kg/day of Control- orAS-ODNs for 14 days. Mice bearing regressed AS-ODN-treated tumors weremonitored up to 4 weeks. A(ii), freshly prepared PANC-1 seed tumorfragments were transplanted into nude mice as above. Infusion with ODNswas initiated two days prior to tumor implantation and continued for anadditional 14 days. These results represent one of three similarexperiments. There were 5 mice for each treatment group. B, AS-ODNtreatment inhibited endogenous tumoral KSR1 expression. Tumoral KSR1 wasimmunoprecipitated from Saline-, Sense- or AS-ODN-treated PANC-1 tumorsand its expression determined by WB as above. C, Inhibition of ksr1 hadno effect of Ras activation in PANC-1 tumors. Ras activation status,measured by the amount of GTP-Ras in Saline-, Control-ODN-, Sense-ODN-or AS-ODN-treated PANC-1 tumors was determined using the Ras activationassay kit. D, KSR AS-ODN treatment prevented A549 tumor growth (i) andinhibited lung metastases via systemic dissemination (ii). A549 seedtumor fragments, freshly prepared as in Methods, were transplanted tonude mice. Treatment with control-or AS-ODNs were initiated when tumorsreached 150 mm³ and continued for additional 18 days (i). When animalswere sacrificed at the end of the experiment, lungs were resected fromcontrol- or AS-ODN-treated mice and stained with Indian ink to visualizesurface lung metastases derived via systemic dissemination (ii). Theseresults represent one of three similar experiments. There were 5 micefor each treatment group.

FIG. 11 depicts a comparative alignment of the mouse KSR polypeptidesequence (SEQ ID NO: 9) and human KSR polypeptide sequence (SEQ ID NO:10).

FIG. 12. A, depicts the nucleic acid coding (cDNA) sequence of mouse ksr(SEQ ID NO: 11). B, depicts the partial nucleic acid coding (cDNA)sequence of human ksr (SEQ ID NO: 12).

FIG. 13 depicts the specific and dose-dependent inhibition of PANC-1cell proliferation by AS-ODN treatment. A, Dose-dependent inhibition ofPANC-1 cell proliferation by AS-ODN treatment; proliferation of K562cells is not inhibited by AS-ODN treatment. B, Western blot analysis ofendogenous KSR1 gene expression in K562 and PANC-1 cells. Treatment ofK562 cells with 5 μM KSR AS-ODN-1 elicited comparable reduction ofendogenous KSR1 gene expression (over 80%) to that observed in PANC-1cells.

FIG. 14 depicts the human KSR1 full length mRNA sequence (SEQ ID NO:24).

FIG. 15 depicts the human KSR nucleic acid and protein sequenceannotated with the locations of the CA1 to CA5 domains and the targetsequences for AS-ODNs. The full length human KSR1 protein is predictedto have 866 amino acids.

FIG. 16 depicts the mouse KSR nucleic acid and protein sequenceannotated with the locations of the CA1 to CA5 domains and the targetsequences for the AS-ODNs.

FIG. 17 depicts the human KSR-1 full length mRNA sequence with thenucleic acid target sequences for human AS-ODN1 through AS-ODN12depicted.

FIG. 18 depicts proliferation assay of PANC-1 cells treated with controlODN, AS-ODN2(181-198) (mouse) (AS-ODN2-old) and AS-ODN2(154-171) (human)(AS-ODN2-new).

FIG. 19 presents percentage of ionizing-radiation induced apoptosis asscored with Annexin V staining in A431 cells expressing vector,wild-type Flag-KSR (KSR-S), Flag-AS-KSR (KSR-AS) or dominant-negativeFlag-Ki-KSR (Ki-KSR).

FIG. 20 presents percentage of ionizing-radiation induced apoptosis asscored with Annexin V staining in A431 cells treated withAS-ODN2(214-231), control ODN or expressing vector.

DETAILED DESCRIPTION

In accordance with the present invention there may be employedconventional molecular biology, microbiology, and recombinant DNAtechniques within the skill of the art. Such techniques are explainedfully in the literature. See, e.g., Sambrook et al, “Molecular Cloning:A Laboratory Manual” (1989); “Current Protocols in Molecular Biology”Volumes I-III [Ausubel, R. M., ed. (1994)]; “Cell Biology: A LaboratoryHandbook” Volumes I-III [J. E. Celis, ed. (1994))]; “Current Protocolsin Immunology” Volumes I-III [Coligan, J. E., ed. (1994)];“Oligonucleotide Synthesis” (M. J. Gait ed. 1984); “Nucleic AcidHybridization” [B. D. Hames & S. J. Higgins eds. (1985)]; “TranscriptionAnd Translation” [B. D. Hames & S.J. Higgins, eds. (1984)]; “Animal CellCulture” [R.I. Freshney, ed. (1986)]; “Immobilized Cells And Enzymes”[IRL Press, (1986)]; B. Perbal, “A Practical Guide To Molecular Cloning”(1984).

Therefore, if appearing herein, the following terms shall have thedefinitions set out below.

The terms “oligonucleotides”, “antisense”, “antisense oligonucleotides”,“KSR ODN”, “KSR antisense” and any variants not specifically listed, maybe used herein interchangeably, and as used throughout the presentapplication and claims refer to nucleic acid material including singleor multiplenucleic acids, and extends to those oligonucleotidescomplementary to the nucleic acid sequences described herein, includingas presented in FIGS. 12A, 12B and 14 and in SEQ ID NOS: 11, 12, and 24,including conserved and activity domains thereof as depicted in FIGS.11, 15 and 16, and having the profile of activities set forth herein andin the Claims, particularly in being capable of inhibiting theexpression of KSR. In particular, the oligonucleotides of the presentinvention may be substantially complementary to nucleic acid sequencespecific to KSR, as provided in SEQ ID NO: 1 or SEQ ID NO:25, or to aportion thereof, as provided for example in SEQ ID NO: 3, 4, 5 and 28.Exemplary oligonucleotides include any of SEQ ID NOS 6-8 and SEQ ID NOS:29-38. Accordingly, nucleic acids or analogs thereof displayingsubstantially equivalent or altered activity are likewise contemplated.These modifications may be deliberate, for example, such asmodifications obtained through site-directed mutagenesis, or may beaccidental, such as those obtained through mutations in hosts that areproducers of the nucleic acids or of KSR.

NH₂ refers to the free amino group present at the amino terminus of apolypeptide. COOH refers to the free carboxy group present at thecarboxy terminus of a polypeptide. In keeping with standard polypeptidenomenclature, J. Biol. Chem., 243:3552-59 (1969), abbreviations foramino acid residues are shown in the following Table of Correspondence:TABLE OF CORRESPONDENCE SYMBOL 1-Letter 3-Letter AMINO ACID Y Tyrtyrosine G Gly glycine F Phe phenylalanine M Met methionine A Alaalanine S Ser serine I Ile isoleucine L Leu leucine T Thr threonine VVal valine P Pro proline K Lys lysine H His histidine Q Gln glutamine EGlu glutamic acid W Trp tryptophan R Arg arginine D Asp aspartic acid NAsn asparagine C Cys cysteine

It should be noted that all amino-acid residue sequences are representedherein by formulae whose left and right orientation is in theconventional direction of amino-terminus to carboxy-terminus.Furthermore, it should be noted that a dash at the beginning or end ofan amino acid residue sequence indicates a peptide bond to a furthersequence of one or more amino-acid residues. The above Table ispresented to conelate the three-letter and one-letter notations whichmay appear alternately herein.

A “replicon” is any genetic element (e.g., plasmid, chromosome, virus)that functions as an autonomous unit of DNA replication in vivo; i.e.,capable of replication under its own control.

A “vector” is a replicon, such as plasmid, phage, virus, retrovirus orcosmid, to which another DNA segment may be attached so as to bringabout the replication of the attached segment.

A “DNA molecule” refers to the polymeric form of deoxyribonucleotides(adenine, guanine, thymine, or cytosine) in its either single strandedform, or a double-stranded helix. This term refers only to the primaryand secondary structure of the molecule, and does not limit it to anyparticular tertiary forms. Thus, this term includes double-stranded DNAfound, inter alia, in linear DNA molecules (e.g., restrictionfragments), viruses, plasmids, and chromosomes. In discussing thestructure of particular double-stranded DNA molecules, sequences may bedescribed herein according to the normal convention of giving only thesequence in the 5′ to 3′ direction along the nontranscribed strand ofDNA (i.e., the strand having a sequence homologous to the mRNA).

An “origin of replication” refers to those DNA sequences thatparticipate in DNA synthesis.

A DNA “coding sequence” is a double-stranded DNA sequence which istranscribed and translated into a polypeptide in vivo when placed underthe control of appropriate regulatory sequences. The boundaries of thecoding sequence are determined by a start codon at the 5′ (amino)terminus and a translation stop codon at the 3′ (carboxyl) terminus. Acoding sequence can include, but is not limited to, prokaryoticsequences, cDNA from eukaryotic mRNA, genomic DNA sequences fromeukaryotic (e.g., mammalian) DNA, and even synthetic DNA sequences. Apolyadenylation signal and transcription termination sequence willusually be located 3′ to the coding sequence.

Transcriptional and translational control sequences are DNA regulatorysequences, such as promoters, enhancers, polyadenylation signals,terminators, and the like, that provide for the expression of a codingsequence in a host cell.

A “promoter sequence” is a DNA regulatory region capable of binding RNApolymerase in a cell and initiating transcription of a downstream (3′direction) coding sequence. For purposes of defining the presentinvention, the promoter sequence is bounded at its 3′ terminus by thetranscription initiation site and extends upstream (5′ direction) toinclude the minimum number of bases or elements necessary to initiatetranscription at levels detectable above background. Within the promotersequence will be found a transcription initiation site (convenientlydefined by mapping with nuclease S1), as well as protein binding domains(consensus sequences) responsible for the binding of RNA polymerase.Eukaryotic promoters will often, but not always, contain “TATA” boxesand “CAT” boxes. Prokaryotic promoters contain Shine-Dalgarno sequencesin addition to the −10 and −35 consensus sequences.

An “expression control sequence” is a DNA sequence that controls andregulates the transcription and translation of another DNA sequence. Acoding sequence is “under the control” of transcriptional andtranslational control sequences in a cell when RNA polymerasetranscribes the coding sequence into mRNA, which is then translated intothe protein encoded by the coding sequence.

A “signal sequence” can be included before the coding sequence. Thissequence encodes a signal peptide, N-terminal to the polypeptide, thatcommunicates to the host cell to direct the polypeptide to the cellsurface or secrete the polypeptide into the media, and this signalpeptide is clipped off by the host cell before the protein leaves thecell. Signal sequences can be found associated with a variety ofproteins native to prokaryotes and eukaryotes.

The term “oligonucleotide,” as used herein in referring to a nucleicacid of the present invention, is defined as a molecule comprised of twoor more ribonucleotides, preferably more than three. Its exact size willdepend upon many factors which, in turn, depend upon the ultimatefunction and use of the oligonucleotide. In particular, and inaccordance with the present invention, the oligonucleotide shouldparticularly associate with the RNA encoding KSR and should be of theappropriate sequence and size or length so as to specifically and stablyassociate with the target RNA such that expression (i.e., translation)of the RNA is blocked or such that stability of the RNA is negativelyaffected. In one particular aspect of the invention, the antisenseoligonucleotides of the present invention are from about 8 to about 50nucleotides in length, particularly oligonucleotides from 10 to 30nucleotides in length, particularly oligonucleotides from 15 to 25nucleotides.

The term “primer” as used herein refers to an oligonucleotide, whetheroccurring naturally as in a purified restriction digest or producedsynthetically, which is capable of acting as a point of initiation ofsynthesis when placed under conditions in which synthesis of a primerextension product, which is complementary to a nucleic acid strand, isinduced, i.e., in the presence of nucleotides and an inducing agent suchas a DNA polymerase and at a suitable temperature and pH. The primer maybe either single-stranded or double-stranded and must be sufficientlylong to prime the synthesis of the desired extension product in thepresence of the inducing agent. The exact length of the primer willdepend upon many factors, including temperature, source of primer anduse of the method. For example, for diagnostic applications, dependingon the complexity of the target sequence, the oligonucleotide primertypically contains 15-25 or more nucleotides, although it may containfewer nucleotides.

The primers herein are selected to be “substantially” complementary todifferent strands of a paiticular target DNA sequence. This means thatthe primers must be sufficiently complementary to hybridize with theirrespective strands. Therefore, the primer sequence need not reflect theexact sequence of the template. For example, a non-complementarynucleotide fragment may be attached to the 5′ end of the primer, withthe remainder of the primer sequence being complementary to the strand.Alternatively, non-complementary bases or longer sequences can beinterspersed into the primer, provided that the primer sequence hassufficient complementarity with the sequence of the strand to hybridizetherewith and thereby form the template for the synthesis of theextension product.

As used herein, the terms “restriction endonucleases” and “restrictionenzymes” refer to bacterial enzymes, each of which cut double-strandedDNA at or near a specific nucleotide sequence.

A cell has been “transformed” by exogenous or heterologous DNA when suchDNA has been introduced inside the cell. The transforming DNA may or maynot be integrated (covalently linked) into chromosomal DNA making up thegenome of the cell. In prokaryotes, yeast, and mammalian cells forexample, the transforming DNA may be maintained on an episomal elementsuch as a plasmid. With respect to eukaryotic cells, a stablytransformed cell is one in which the transforming DNA has becomeintegrated into a chromosome so that it is inherited by daughter cellsthrough chromosome replication. This stability is demonstrated by theability of the eukaryotic cell to establish cell lines or clonescomprised of a population of daughter cells containing the transformingDNA. A “clone” is a population of cells derived from a single cell orcommon ancestor by mitosis. A “cell line” is a clone of a primary cellthat is capable of stable growth in vitro for many generations.

Two DNA sequences are “substantially homologous” when at least about 75%(preferably at least about 80%, and most preferably at least about 90 or95%) of the nucleotides match over the defined length of the DNAsequences. Sequences that are substantially homologous can be identifiedby comparing the sequences using standard software available in sequencedata banks, or in a Southern hybridization experiment under, forexample, stringent conditions as defined for that particular system.Defining appropriate hybridization conditions is within the skill of theart. See, e.g., Maniatis et al., supra; DNA Cloning, Vols. I & II,supra; Nucleic Acid Hybridization, supra.

It should be appreciated that also within the scope of the presentinvention are DNA or nucleic acid sequences which code for KSR havingthe same amino acid sequence as the KSR sequences disclosed herein,including SEQ ID NO: 11 and SEQ ID NO: 12, but which are degeneratethese sequences. By “degenerate to” is meant that a differentthree-letter codon is used to specify a particular amino acid. It iswell known in the art that the following codons can be usedinterchangeably to code for each specific amino acid: Phenylalanine (Pheor F) UUU or UUC Leucine (Leu or L) UUA or UUG or CUU or CUC or CUA orCUG Isoleucine (Ile or I) AUU or AUC or AUA Methionine (Met or M) AUGValine (Val or V) GUU or GUC of GUA or GUG Serine (Ser or S) UCU or UCCor UCA or UCG or AGU or AGC Proline (Pro or P) CCU or CCC or CCA or CCGThreonine (Thr or T) ACU or ACC or ACA or ACG Alanine (Ala or A) GCU orGCG or GCA or GCG Tyrosine (Tyr or Y) UAU or UAC Histidine (His or H)CAU or CAC Glutamine (Gln or Q) CAA or CAG Asparagine (Asn or N) AAU orAAC Lysine (Lys or K) AAA or AAG Aspartic Acid (Asp or D) GAU or GACGlutamic Acid (Glu or E) GAA or GAG Cysteine (Cys or C) UGU or UGCArginine (Arg or R) CGU or CGC or CGA or CGG or AGA or AGG Glycine (Glyor G) GGU or GGC or GGA or GGG Tryptophan (Trp or W) UGG Terminationcodon UAA (ochre) or UAG (amber) or UGA (opal)

It should be understood that the codons specified above are for RNAsequences. The corresponding codons for DNA have a T substituted for U.

Mutations can be made in ksr such that a particular codon is changed toa codon which codes for a different amino acid. Such a mutation isgenerally made by making the fewest nucleotide changes possible. Asubstitution mutation of this sort can be made to change an amino acidin the resulting protein in a non-conservative manner (i.e., by changingthe codon from an amino acid belonging to a grouping of amino acidshaving a particular size or characteristic to an amino acid belonging toanother grouping) or in a conservative manner (i.e., by changing thecodon from an amino acid belonging to a grouping of amino acids having aparticular size or characteristic to an amino acid belonging to the samegrouping). Such a conservative change generally leads to less change inthe structure and function of the resulting protein. A non-conservativechange is more likely to alter the structure, activity or function ofthe resulting protein. The present invention should be considered toinclude sequences containing conservative changes which do notsignificantly alter the activity or binding characteristics of theresulting protein.

The following is one example of various groupings of amino acids:

Amino Acids with Nonpolar R Groups

Alanine, Valine, Leucine, Isoleucine, Proline, Phenylalanine,Tryptophan, Methionine

Amino Acids with Uncharged Polar R Groups

Glycine, Serine, Threonine, Cysteine, Tyrosine, Asparagine, Glutamine

Amino Acids with Charged Polar R Groups (Negatively Charged at Ph 6.0)

Aspartic Acid, Glutamic Acid

Basic Amino Acids (Positively Charged at pH 6.0)

Lysine, Arginine, Histidine (at pH 6.0)

Another grouping may be those amino acids with phenyl groups:

Phenylalanine, Tryptophan, Tyrosine

Another grouping may be according to molecular weight (i.e., size of Rgroups): Glycine 75 Alanine 89 Serine 105 Proline 115 Valine 117Threonine 119 Cysteine 121 Leucine 131 Isoleucine 131 Asparagine 132Aspartic acid 133 Glutamine 146 Lysine 146 Glutamic acid 147 Methionine149 Histidine (at pH 6.0) 155 Phenylalanine 165 Arginine 174 Tyrosine181 Tryptophan 204

Particularly preferred substitutions are:

-   -   Lys for Arg and vice versa such that a positive charge may be        maintained;    -   Glu for Asp and vice versa such that a negative charge may be        maintained;    -   Ser for Thr such that a free —OH can be maintained; and    -   Gln for Asn such that a free NH₂ can be maintained.

Amino acid substitutions may also be introduced to substitute an aminoacid with a particularly preferable property. For example, a Cys may beintroduced a potential site for disulfide bridges with another Cys. AHis may be introduced as a particularly “catalytic” site (i.e., His canact as an acid or base and is the most common amino acid in biochemicalcatalysis). Pro may be introduced because of its particularly planarstructure, which induces β-turns in the protein's structure.

Two amino acid sequences are “substantially homologous” when at leastabout 70% of the amino acid residues (preferably at least about 80%, andmost preferably at least about 90 or 95%) are identical, or representconservative substitutions.

A “heterologous” region of the DNA construct is an identifiable segmentof DNA within a larger DNA molecule that is not found in associationwith the larger molecule in nature. Thus, when the heterologous regionencodes a mammalian gene, the gene will usually be flanked by DNA thatdoes not flank the mammalian genomic DNA in the genome of the sourceorganism. Another example of a heterologous coding sequence is aconstruct where the coding sequence itself is not found in nature (e.g.,a cDNA where the genomic coding sequence contains introns, or syntheticsequences having codons different than the native gene). Allelicvariations or naturally-occurring mutational events do not give rise toa heterologous region of DNA as defined herein.

The phrase “pharmaceutically acceptable” refers to molecular entitiesand compositions that are physiologically tolerable and do not typicallyproduce an allergic or similar untoward reaction, such as gastric upset,dizziness and the like, when administered to a human.

The phrase “therapeutically effective amount” is used herein to mean anamount sufficient to prevent, and preferably reduce by at least about 30percent, more preferably by at least 50 percent, more preferably by atleast 70 percent, most preferably by at least 90 percent, a clinicallysignificant change in the mitotic activity of a target cellular mass, orother feature of pathology such as for example, reduced tumor mass,reduced tumor cell proliferation, reduction in metastatic capacity, orenhanced apoptosis, as may attend its presence and activity.

As used herein, “pg” means picogram, “ng” means nanogram, “ug” or “μg”mean microgram, “mg” means milligram, “ul” or “μl” mean microliter, “ml”means milliliter, “l” means liter.

A DNA sequence is “operatively linked” to an expression control sequencewhen the expression control sequence controls and regulates thetranscription and translation of that DNA sequence. The term“operatively linked” includes having an appropriate start signal (e.g.,ATG) in front of the DNA sequence to be expressed and maintaining thecorrect reading frame to permit expression of the DNA sequence under thecontrol of the expression control sequence and production of the desiredproduct encoded by the DNA sequence. If a gene that one desires toinsert into a recombinant DNA molecule does not contain an appropriatestart signal, such a start signal can be inserted in front of the gene.

The term “standard hybridization conditions” refers to salt andtemperature conditions substantially equivalent to 5×SSC and 65° C. forboth hybridization and wash. However, one skilled in the art willappreciate that such “standard hybridization conditions” are dependenton particular conditions including the concentration of sodium andmagnesium in the buffer, nucleotide sequence length and concentration,percent mismatch, percent formamide, and the like. Also important in thedetermination of “standard hybridization conditions” is whether the twosequences hybridizing are RNA-RNA, DNA-DNA or RNA-DNA. Such standardhybridization conditions are easily determined by one skilled in the artaccording to well known formulae, wherein hybridization is typically10-20° C. below the predicted or determined T_(m) with washes of higherstringency, if desired.

The present invention relates to methods and compositions for thespecific inhibition of expression and/or activity of kinase suppressorof Ras (KSR). In particular, the invention provides genetic approachesand nucleic acids for the specific inhibition of KSR. It is hereindemonstrated that on specific inhibition of KSR the Ras pathway isdisrupted and, specifically, that Ras-mediated tumors, tumorigenesis andmetastasis regress, are inhibited, or are blocked. In particularantisense oligonucleotides and the expression of nucleic acidcomplementary to KSR RNA specifically inhibits expression of KSR andblocks gƒRas mediated tumorigenesis.

The present invention provides an oligonucleotide which is substantiallycomplementary to a region of KSR RNA, wherein said oligonucleotideinhibits the expression of KSR. The invention further provides anoligonucleotide which is substantially complementary to a nucleic acidencoding mammalian KSR. In a particular embodiment, an oligonucleotideis provided which is substantially complementary to a nucleic acidencoding human KSR.

In an aspect of the invention an oligonucleotide is provided which issubstantially complementary to a translation initiation site, 5′untranslated region, coding region or 3′ untranslated region of mRNAencoding mammalian KSR. In one such aspect, the invention thus providesan oligonucleotide which is substantially complementary to a translationinitiation site, 5′ untranslated region, coding region or 3′untranslated region of mRNA encoding human KSR as provided in FIG. 14and SEQ ID NO: 24. In a particular embodiment, the invention provides anantisense oligonucleotide substantially complementary to the N-terminalcoding region of mammalian KSR mRNA, particularly to the region ofnucleotides 1 through 761 of the coding region of mammalian KSR mRNA. Inone embodiment, the invention includes an antisense oligonucleotidecomprising a sequence substantially complementary to the CA1 region ofKSR (SEQ ID NO:1 and SEQ ID NO:25). The invention providesoligonucleotides comprising a sequence substantially complementary tonucleotides encoding amino acids 33 to 72 of human KSR (SEQ ID NO:26) or42 to 82 of mouse KSR (amino acids SEQ ID NO: 2), or a portion thereof,of the sequence of KSR.

In a further embodiment, the invention includes an antisenseoligonucleotide comprising a sequence substantially complementary tonucleotides 97 to 216 of the coding sequence of human KSR (SEQ ID NO:25)corresponding to nucleotides 124 to 243 of mouse KSR (SEQ ID NO: 1), ora portion thereof. The invention provides an oligonucleotide comprisinga sequence substantially complementary to nucleotides encoding aminoacids 33 to 72 of human KSR (SEQ ID NO:26) and 42 to 82 of mouse KSR(SEQ ID NO: 2) or a portion thereof. In particular, oligonucleotides ofthe invention include oligonucleotides comprising a sequencesubstantially complementary to nucleotides selected from the group of:nucleotides 124 to 141 of human KSR, corresponding to nucleotides 151 to168 of mouse KSR (SEQ ID NO: 3); nucleotides 154 to 171 of human KSR(SEQ ID NO: 27), corresponding closely to nucleotides 181 to 189 ofmouse KSR (SEQ ID NO: 4), with a single base change at the 5′-mostnucleotide; and nucleotides 187 to 204 of human KSR, corresponding tonucleotides 214 to 231 of mouse KSR (SEQ ID NO: 5). The inventionincludes antisense oligonucleotides comprising a sequence selected fromthe group of SEQ ID NOS: 6-8 and SEQ ID NOS: 29-38.

In a particular aspect, the nucleic acids and oligonucleotides of thepresent invention may be modified, either by manipulation of thechemical backbone of the nucleic acids or by covalent or non-covalentattachment of other moieties. In each or any case, such manipulation orattachment may serve to modify the stability, cellular, tissue or organuptake, or otherwise enhance efficacy of the nucleic acids andcovalently linked to other molecules, including but not limited topolypeptides, carbohydrates, lipid or lipid-like moieties, ligands,chemical agents or compounds, which may serve to enhance the uptake,stability or to target the oligonucleotides.

In further embodiments, the oligonucleotides of the present inventionare modified in their chemical backbone. In a particular embodiment, theoligonucleotides comprise at least one phosphorothioate linkage.

The oligonucleotides of the present invention may be combined witholigonucleotides directed to other targets, by mixture or bynon-covalent or covalent attachment. For instance, the KSR antisenseoligonucleotides of the present invention may be combined with antisensedirected to raf as described in U.S. Pat. No. 6,391,636 (incorporatedherein by reference) or to other oncogenic or proliferative proteins.

Recombinant DNA molecules comprising a nucleic acid sequence whichencodes on transcription an antisense RNA complementary to mammalian KSRRNA or a portion thereof are provided by the invention. Further, therecombinant DNA molecules comprise a nucleic acid sequence wherein saidnucleic acid sequence is operatively linked to a transcription controlsequence. Cell lines transfected with these recombinant DNA moleculesare also included in the invention.

In a further aspect, an expression vector is provided which is capableof expressing a nucleic acid which is substantially complementary to thecoding sequence of KSR RNA, or a portion thereof, wherein said nucleicacid inhibits the expression of KSR. In a particular aspect, thisincludes an expression vector capable of expressing an oligonucleotidewhich is substantially complementary to the CA1 region of the codingsequence of KSR RNA, or a portion thereof, wherein said oligonucleotideinhibits the expression of KSR.

In an additional embodiment, the invention provides a compositioncomprising an expression vector and a pharmaceutically acceptablecarrier or diluent, wherein said expression vector is capable ofexpressing nucleic acid which is substantially complementary to thecoding sequence of KSR RNA, or a portion thereof, wherein said nucleicacid inhibits the expression of KSR.

Methods for inhibiting expression of KSR are provided. In one aspect, amethod of inhibiting the expression of mammalian KSR comprisingcontacting cells which express KSR with an effective amount of a nucleicacid which is complementary to a portion of the mRNA encoding KSR isincluded. In particular, a method of inhibiting the expression ofmammalian KSR is provided, comprising contacting cells which express KSRwith an effective amount of the oligonucleotide of the present inventionwhereby expression of mammalian KSR is inhibited.

In addition, a method is provided for identifying compounds or agentswhich inhibit the expression of KSR comprising the steps of:

-   -   (a) incubating a cell expressing KSR in the presence and absence        of a candidate compound or agent; and    -   (b) detecting or measuring the expression of KSR in the presence        and absence of a candidate compound or agent,        whereby a decrease in the expression of KSR in the presence of        said candidate compound or agent versus in the absence of said        candidate compound or agent indicates that said compound or        agent inhibits the expression of KSR.

Methods for inhibiting activity, including and preferably kinase orphosphorylation activity, of KSR are provided. In one aspect, a methodof inhibiting the activity of mammalian KSR comprising contacting cellswhich express KSR with an effective amount of a compound or agent thatinhibits or blocks KSR is included. In particular, a method ofinhibiting the activity of mammalian KSR is provided, comprisingcontacting cells which express KSR with an effective amount of acompound, agent or composition of the present invention whereby activityof mammalian KSR is inhibited.

In addition, a method is provided for identifying compounds or agentswhich inhibit the activity, including kinase or phosphorylationactivity, of KSR comprising the steps of:

-   -   (a) incubating a cell expressing KSR in the presence and absence        of a candidate compound or agent; and    -   (b) detecting or measuring the activity of KSR in the presence        and absence of a candidate compound or agent,        whereby a decrease in the activity of KSR in the presence of        said candidate compound or agent versus in the absence of said        candidate compound or agent indicates that said compound or        agent inhibits the activity of KSR. In one aspect of the        invention the activity and/or expression of KSR may be assessed        or monitored by determining the phosphorylation status of the        KSR kinase target or of a peptide comprising a kinase target        sequence domain. For instance, the phosphorylation status of Raf        or a Raf-derived peptide may be utilized in such an assessment.        The activity or expression of KSR may also be assessed in vitro        or in vivo in tumor cells or tumor animal models, whereby Ras        mediated tumorigenesis, cell proliferation or metastasis is        monitored, including as described in the Examples herein.

The present invention naturally contemplates several means forpreparation of the nucleic acids and oligonucleotides of the presentinvention, including as illustrated herein known recombinant techniques,and the invention is accordingly intended to cover such syntheticpreparations within its scope. The knowledge of the cDNA and amino acidsequences of KSR as disclosed herein facilitates the preparation of thenucleic acids of the invention by such recombinant techniques, andaccordingly, the invention extends to expression vectors prepared fromthe disclosed DNA sequences for expression in host systems byrecombinant DNA techniques, and to the resulting transformed hosts.

Another feature of this invention is the expression of the nucleic acidsdisclosed herein. As is well known in the art, nucleic acid or DNAsequences may be expressed by operatively linking them to an expressioncontrol sequence in an appropriate expression vector and employing thatexpression vector to transform an appropriate unicellular host. Suchoperative linking of a nucleic acid sequence of this invention to anexpression control sequence, of course, includes, if not already part ofthe DNA sequence, the provision of an initiation codon, ATG, in thecorrect reading frame upstream of the DNA sequence.

A wide variety of host/expression vector combinations may be employed inexpressing the DNA sequences of this invention. Useful expressionvectors, for example, may consist of segments of chromosomal,non-chromosomal and synthetic DNA sequences. Suitable vectors includederivatives of SV40 and known bacterial plasmids, e.g., E. coli plasmidscol E1, pCR1, pBR322, pMB9 and their derivatives, plasmids such as RP4;phage DNAS, e.g., the numerous derivatives of phage λ, e.g., NM989, andother phage DNA, e.g., M13 and filamentous single stranded phage DNA;yeast plasmids such as the 2μ plasmid or derivatives thereof; vectorsuseful in eukaryotic cells, such as vectors useful in insect ormammalian cells; vectors derived from combinations of plasmids and phageDNAs, such as plasmids that have been modified to employ phage DNA orother expression control sequences; and the like. In addition, viral andretroviral vectors, including but not limited to adenovirus andadeno-associated virus, may be useful in such expression.

Any of a wide variety of expression control sequences—sequences thatcontrol the expression of a DNA sequence operatively linked to it—may beused in these vectors to express the DNA sequences of this invention.Such useful expression control sequences include, for example, the earlyor late promoters of SV40, CMV, vaccinia, polyoma or adenovirus, the lacsystem, the trp system, the TAC system, the TRC system, the LTR system,the major operator and promoter regions of phage λ, the control regionsof fd coat protein, the promoter for 3-phosphoglycerate kinase or otherglycolytic enzymes, the promoters of acid phosphatase (e.g., Pho5), thepromoters of the yeast-mating factors, and other sequences known tocontrol the expression of genes of prokaryotic or eukaryotic cells ortheir viruses, and various combinations thereof.

A wide variety of unicellular host cells are also useful in expressingthe DNA sequences of this invention. These hosts may include well knowneukaryotic and prokaryotic hosts, such as strains of E. coli,Pseudomonas, Bacillus, Streptomyces, fungi such as yeasts, and animalcells, such as CHO, R1.1, B-W and L-M cells, African Green Monkey kidneycells (e.g., COS 1, COS 7, BSC1, BSC40, and BMT10), insect cells (e.g.,Sf9), and tumor cells, transformed cells, human cells and plant cells intissue culture.

It will be understood that not all vectors, expression control sequencesand hosts will function equally well to express the DNA sequences ofthis invention. Neither will all hosts function equally well with thesame expression system. However, one skilled in the art will be able toselect the proper vectors, expression control sequences, and hostswithout undue experimentation to accomplish the desired expressionwithout departing from the scope of this invention. For example, inselecting a vector, the host must be considered because the vector mustfunction in it. The vector's copy number, the ability to control thatcopy number, and the expression of any other proteins encoded by thevector, such as antibiotic markers, will also be considered.

In selecting an expression control sequence, a variety of factors willnormally be considered. These include, for example, the relativestrength of the system, its controllability, and its compatibility withthe particular DNA sequence or gene to be expressed, particularly asregards potential secondary structures. Suitable unicellular hosts willbe selected by consideration of, e.g., their compatibility with thechosen vector, their secretion characteristics, their ability to foldproteins conectly, and their fermentation requirements, as well as thetoxicity to the host of the product encoded by the DNA sequences to beexpressed, and the ease of purification of the expression products.Considering these and other factors a person skilled in the art will beable to construct a variety of vector/expression control sequence/hostcombinations that will express the DNA sequences of this invention onfermentation or in large scale animal culture.

The present invention further includes transgenic animals and animalmodels wherein KSR is knocked out or otherwise nullified (as in theksr^(−/−) animals described herein) or wherein KSR is overexpressed asfurther described herein. Such animal models include mammals forinstance mice, rats, pigs, rabbits, dogs, monkeys, etc. and any otherrecognized vertebrate or invertebrate system for study, including ducks,fish, drosphila, C. elegans, etc. In the case of nullified KSR, theseanimals are useful for the study of oncogenesis or the blockage oftumorigenesis in a KSR null background, including to identify otherfactors in tumorigenesis or metastasis. In the case of KSRoverexpressors, wherein tumorigenesis, cell proliferation and metastasisis enhanced, these systems may be useful for the rapid study of tumormodels and for evaluating potential anti-cancer compounds or agents,including those targeting KSR as well as other pathways.

As mentioned above, nucleic acids and oligonucleotides of the presentinvention can be prepared synthetically rather than cloned. In general,one will select preferred codons for the intended host if the sequencewill be used for expression. The complete sequence is assembled fromoverlapping oligonucleotides prepared by standard methods and assembledinto a complete coding sequence. See, e.g., Edge, Nature, 292:756(1981); Nambair et al., Science, 223:1299 (1984); Jay et al., J. Biol.Chem., 259:6311 (1984).

Antisense nucleic acids are DNA or RNA molecules that are complementaryto at least a portion of a specific mRNA molecule. (See Weintraub, 1990;Marcus-Sekura, 1988.) In the cell, they hybridize to that mRNA, forminga double stranded molecule and interfering with the expression of mRNAinto protein. Antisense methods have been used to inhibit the expressionof many genes in vitro (Marcus-Sekura, 1988; Hambor et al., 1988).

The antisense oligonucleotides of the invention are selected assubstantially complementary to a region of KSR mRNA. Theoligonucleotides of the present invention may be complementary toregions including but not limited to: a) the 5′-cap site of an mRNAmolecule (Ojala et al. (1997) Antisense Nucl. Drug Dev. 7:31-38); b) thetranscription start site (Monia et al. (1992) J. Biol. Chem.267:19954-19962); c) the translation initiation codon (Dean et al.(1994) Proc. Natl. Acad. Sci. U.S.A. 91:11762-11766); d) the translationstop codon (Wang et al. (1995) Proc. Natl. Acad. Sci. USA 92:3318-3322);e) mRNA splice sites (Agrawal et al. (1988) Proc. Natl. Acad. Sci.U.S.A. 86:7790-7794; Colige et al. (1993) Biochem. 32:7-11); f) the5′-untranslated region of mRNA molecules (Duff et al. (1995) J. Biol.Chem. 270:7161-7166; Yamagami et al. (1996) Blood 87:2878-2884); g) the3′-untranslated region of mRNA molecules (Bennett et al. (1994) J.Immunol. 152:3530-3540; Dean et al. (1994) J. Biol. Chem.269:16146-16424); and h) the coding region (Laptev et al. (1994)Biochem. 33:11033-11039; Yamagami et al. (1996) Blood 87:2878-2884).

The skilled artisan can readily utilize any of several strategies tofacilitate and simplify the selection process for nucleic acids andoligonucleotides effective in inhibition of KSR expression. Predictionsof the binding energy or calculation of thermodynamic indices between anolionucleotide and a complementary sequence in an mRNA molecule may beutilized (Chiang et al. (1991) J. Biol. Chem. 266:18162-18171; Stull etal. (1992) Nucl. Acids Res. 20:3501-3508). Antisense oligonucleotidesmay be selected on the basis of secondary structure (Wickstrom et al(1991) in Prospects for Antisense Nucleic Acid Therapy of Cancer andAIDS, Wickstrom, ed., Wiley-Liss, Inc., New York, pp. 7-24; Lima et al.(1992) Biochem. 31:12055-12061). Schmidt and Thompson (U.S. Pat. No.6,416,951) describe a method for identifying a functional antisenseagent comprising hybridizing an RNA with an oligonucleotide andmeasuring in real time the kinetics of hybridization by hybridizing inthe presence of an intercalation dye or incorporating a label andmeasuring the spectroscopic properties of the dye or the label's signalin the presence of unlabelled oligonucleotide. In addition, any of avariety of computer programs may be utilized which predict suitableantisense oligonucleotide sequences or antisense targets utilizingvarious criteria recognized by the skilled artisan, including forexample the absence of self-complementarity, the absence hairpin loops,the absence of stable homodimer and duplex formation (stability beingassessed by predicted energy in kcal/mol). Examples of such computerprograms are readily available and known to the skilled artisan andinclude the OLIGO 4 or OLIGO 6 program (Molecular Biology Insights,Inc., Cascade, Colo.) and the Oligo Tech program (Oligo TherapeuticsInc., Wilsonville, Oreg.).

In addition, antisense oligonucleotides suitable in the presentinvention may be identified by screening an oligonucleotide library, ora library of nucleic acid molecules, under hybridization conditions andselecting for those which hybridize to the target RNA or nucleic acid(see for example U.S. Pat. No. 6,500,615). Mishra and Toulme have alsodeveloped a selection procedure based on selective amplification ofoligonucleotides that bind target (Mishra et al (1994) Life Sciences317:977-982). Oligonucleotides may also be selected by their ability tomediate cleavage of target RNA by RNAse H, by selection andcharacterization of the cleavage fragments (Ho et al (1996) Nucl AcidsRes 24:1901-1907; Ho et al (1998) Nature Biotechnology 16:59-630).Generation and targeting of oligonucleotides to GGGA motifs of RNAmolecules has also been described (U.S. Pat. No. 6,277,981).

Inhibition of ksr gene expression can be measured in ways which areroutine in the art, for example by Northern blot assay of mRNAexpression or Western blot assay of protein expression as well known tothe skilled artisan. Effects on cell proliferation or tumor cell growthcan also be measured, in vitro or in vivo, in cell, tumor or animalmodel systems, by methods well known to the skilled artisan, includingas taught in the examples of the instant application. Similarly,inhibition of KSR activity, particularly phosphorylation or kinaseactivity may be measured.

“Substantially complementary” is used to indicate a sufficient degree ofcomplementarity such that stable and specific binding occurs between theDNA or RNA target and the oligonucleotide or nucleic acid. It isunderstood that an oligonucleotide need not be 100% complementary to itstarget nucleic acid sequence to be specifically hybridizable. Anoligonucleotide is specifically hybridizable when binding of theoligonucleotide to the target interferes with the normal function of thetarget molecule to cause a loss of utility or expression, and there is asufficient degree of complementarity to avoid non-specific binding ofthe oligonucleotide to non-target sequences under physiologicalconditions in the case of in vivo assays or therapeutic treatment or, inthe case of in vitro assays, under conditions in which the assays areconducted.

In the context of this invention, the term “oligonucleotide” refers toan oligomer or polymer of nucleotide or nucleoside monomers consistingof naturally occurring bases, sugars and intersugar (backbone) linkages.Oligonucleotide includes oligomers comprising non-naturally occurringmonomers, or portions thereof, which function similarly and suchmodified or substituted oligonucleotides may be preferred over nativeforms because of, for example, enhanced cellular uptake and increasedstability against nucleases. The oligonucleotides of the presentinvention may contain two or more chemically distinct regions, each madeup of at least one nucleotide, for instance, at least one region ofmodified nucleotides that confers one or more beneficial properties (forexample, increased nuclease resistance, increased uptake into cells,increased binding affinity for the RNA target) and a region that is asubstrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids(for example, RNase H—a cellular endonuclease which cleaves the RNAstrand of an RNA:DNA duplex).

In a preferred embodiment, the region of the oligonucleotide which ismodified to increase KSR mRNA binding affinity comprises at least onenucleotide modified at the 2′ position of the sugar, most preferably a2′-O-alkyl, 2′-O-alkyl-O-alkyl or 2′-fluoro-modified nucleotide. Suchmodifications are routinely incorporated into oligonucleotides and theseoligonucleotides have been shown to have a higher Tm (i.e., highertarget binding affinity) than 2′-deoxyoligonucleotides against a giventarget. In another preferred embodiment, the oligonucleotide is modifiedto enhance nuclease resistance. Cells contain a variety of exo- andendo-nucleases which can degrade nucleic acids. A number of nucleotideand nucleoside modifications have been shown to confer relativelygreater resistance to nuclease digestion. Oligonucleotides which containat least one phosphorothioate modification are presently more preferred(Geary, R. S. et al (1997) Anticancer Drug Des 12:383-93; Henry, S. P.et al (1997) Anticancer Drug Des 12:395-408; Banerjee, D. (2001) CurrOpin Investig Drugs 2:574-80). In some cases, oligonucleotidemodifications which enhance target binding affinity are also,independently, able to enhance nuclease resistance.

Specific examples of some preferred oligonucleotides envisioned for thisinvention include those containing modified backbones, for example,phosphorothioates, phosphotriesters, methyl phosphonates, short chainalkyl or cycloalkyl intersugar linkages or short chain heteroatomic orheterocyclic intersugar linkages. Most preferred are oligonucleotideswith phosphorothioate backbones and those with heteroatom backbones. Theamide backbones disclosed by De Mesmaeker et al. (1995) Acc. Chem. Res.28:366-374) are also preferred. Also preferred are oligonucleotideshaving morpholino backbone structures (Summerton and Weller, U.S. Pat.No. 5,034,506). In other preferred embodiments, such as the peptidenucleic acid (PNA) backbone, the phosphodiester backbone of theoligonucleotide is replaced with a polyamide backbone, the nucleobasesbeing bound directly or.indirectly to the aza nitrogen atoms of thepolyamide backbone (Nielsen et al., Science, 1991, 254, 1497).Oligonucleotides may also contain one or more substituted sugarmoieties. Preferred oligonucleotides comprise one of the following atthe 2′ position: OH, SH, SCH₃, F, OCN, heterocycloalkyl;heterocycloalkaryl; aminoalkylamino; polyalkylamino; substituted silyl;an RNA cleaving group; a reporter group; an intercalator; a group forimproving the pharmacokinetic properties of an oligonucleotide; or agroup for improving the pharmacodynamic properties of an oligonucleotideand other substituents having similar properties. Similar modificationsmay also be made at other positions on the oligonucleotide, particularlythe 3′ position of the sugar on the 3′ terminal nucleotide and the 5′position of 5′ terminal nucleotide.

Oligonucleotides may also include, additionally or alternatively basemodifications or substitutions. As used herein, “unmodified” or“natural” nucleobases include adenine (A), guanine (G), thymine (T),cytosine (C) and uracil (U). Modified nucleobases include nucleobasesfound only infrequently or transiently in natural nucleic acids, e.g.,hypoxanthine, 6-methyladenine, 5-me pyrimidines, particularly5-methylcytosine (5-me-C) (Sanghvi, Y. S., in Crooke, S. T. and Lebleu,B., eds., Antisense Research and Applications, CRC Press, Boca Raton,1993, pp. 276-278), 5-hydroxymethylcytosine (HMC), glycosyl HMC andgentobiosyl HMC, as well as synthetic nucleobases, including but notlimited to, 2-aminoadenine, 2-thiouracil, 2-thiothymine, 5-bromouracil,5-hydroxymethyluracil, 8-azaguanine, 7-deazaguanine (Kornberg, A., DNAReplication, W. H. Freeman & Co., San Francisco, 1980, pp75-77;Gebeyehu, G., et al., 1987, Nucl. Acids Res. 15:4513). A “universal”base known in the art, e.g., inosine, may be included.

Another modification of the oligonucleotides of the invention involveschemically linking to the oligonucleotide one or more moieties orconjugates which enhance the activity or cellular uptake of theoligonucleotide. Such moieties include but are not limited to lipidmoieties such as a cholesterol moiety, a cholesteryl moiety (Letsingeret al. (1989) Proc. Natl. Acad. Sci. USA 86: 6553), cholic acid(Manoharan et al. (1994) Bioorg. Med. Chem. Let. 4:1053), a thioether,for example, hexyl-S-tritylthiol (Manoharan et al. (1992) Ann. N.Y.Acad. Sci. 660: 306; Manoharan et al. (1993) Bioorg. Med. Chem. Let. 3:2765), a thiocholesterol (Oberhauser et al. (1992) Nucl. AcidsRes.20:533), an aliphatic chain, for example, dodecandiol or undecylresidues (Saison-Behmoaras et al. (1991) EMBO J. 10:111; Kabanov et al.(1990) FEBS Lett. 259:327; Svinarchuk et al. (1993) Biochimie 75:49), aphospholipid, a polyamine or a polyethylene glycol chain (Manoharan etal. (1995) Nucleosides & Nucleotides 14:969). Oligonucleotidescomprising lipophilic moieties, and methods for preparing sucholigonucleotides are known in the art, for example, U.S. Pat. Nos.5,138,045, 5,218,105 and 5,459,255.

Farrel and Kloster (U.S. Pat. No. 6,310,047) describe the enhancement ofdelivery and of in vivo nuclease resistance of antisenseoligonucleotides using high affinity DNA binding polynuclear platinumcompounds.

It is not necessary for all positions in a given oligonucleotide to beuniformly modified, and more than one of the aforementionedmodifications may be incorporated in a single oligonucleotide or even ata single nucleoside within an oligonucleotide.

The oligonucleotides in accordance with this invention preferably arefrom about 8 to about 50 nucleotides in length. Particularly prefelTedoligonucleotides are from 10 to 30 nucleotides in length, particularlypreferred are from 15 to 25 nucleotides. In the context of thisinvention it is understood that this encompasses non-naturally occurringoligomers as hereinbefore described, having 8 to 50 monomers.

The oligonucleotides used in accordance with this invention may beconveniently and routinely made through the well-known technique ofsolid phase synthesis. Equipment for such synthesis is sold by severalvendors including Applied Biosystems. Any other means for such synthesismay also be employed; the actual synthesis of the oligonucleotides iswell within the talents of the skilled artisan. It is also well known touse similar techniques to prepare other oligonucleotides such as thephosphorothioates and alkylated derivatives.

The therapeutic possibilities that are raised by the methods andcompositions, particularly oligonucleotides, of the present inventionderive from the demonstration in the Examples herein that inactivationof KSR, including by genetic knockout, by inhibition of its expressionutilizing antisense oligonucleotides, and by expression of reversecomplement RNA or antisense DNA constructs, results in specific blockageof Ras-mediated tumorigenesis and cellular hyperproliferation, includingvia gƒ-Ras, and treatment or inhibition of progression of cancer. Thepresent invention contemplates pharmaceutical intervention in thecascade of reactions in which overexpressed or amplified, hyperactivatedor oncogenic Ras, including gƒ-Ras, is implicated, to regress, block,treat or inhibit the progression of Ras-mediated tumors, oncogenesis andmetastasis. Thus, in instances where it is desired to reduce or inhibitRas, including gƒ-Ras, the nucleic acids and oligonucleotides of thepresent invention could be introduced to block or inhibit the Raspathway.

The invention further includes a method of treating or preventing ahyperproliferative condition associated with the expression of gƒ-Ras orheightened expression or hyperactivation of Ras or the Ras pathway in amammal comprising administering to said mammal a therapeuticallyeffective amount of a compound or agent which inhibits the expression oractivity of mammalian KSR protein. In one aspect of this method, saidcompound or agent is an antisense oligonucleotide which specificallyhybridizes to a portion of the mRNA encoding KSR.

A method of treating or preventing a hyperproliferative conditionassociated with the expression of gƒ-Ras or heightened expression orhyperactivation of Ras in a mammal is provided, comprising expressing insaid mammal or administering to said mammal a therapeutically effectiveamount of a nucleic acid which is complementary to a portion of the mRNAencoding KSR.

In a further aspect, a method of treating or inhibiting the progressionof cancer in a mammal is included, comprising administering to a mammala therapeutically effective amount of a compound or agent which inhibitsthe expression or activity of mammalian KSR protein. Cancers which aresusceptible to the invention's method include cancer selected from thegroup of pancreatic cancer, lung cancer, skin cancer, urinary tractcancer, bladder cancer, liver cancer, thyroid cancer, colon cancer,intestinal cancer, leukemia, lymphoma, neuroblastoma, head and neckcancer, breast cancer, ovarian cancer, stomach cancer, esophageal cancerand prostate cancer.

Thus, a method is provided for treating or inhibiting the progression ofcancer in a mammal comprising administering to a mammal atherapeutically effective amount of one or more oligonucleotide of thepresent invention.

The present invention further contemplates therapeutic compositionsuseful in practicing the therapeutic methods of this invention. Asubject therapeutic composition includes, in admixture, apharmaceutically acceptable carrier (excipient) or diluent and one ormore nucleic acid or oligonucleotide of the invention as describedherein as an active ingredient. In a preferred embodiment, thecomposition comprises an oligonucleotide capable of inhibiting theexpression of KSR.

Compositions of the nucleic acids and oligonucleotides are an additionalaspect of the invention. The invention includes a composition comprisingan oligonucleotide which is substantially complementary to a region ofKSR RNA and a pharmaceutically acceptable carrier or diluent. Theinvention thus provides a pharmaceutical composition comprising atherapeutically effective amount of an oligonucleotide which issubstantially complementary to a region of KSR RNA and apharmaceutically acceptable carrier or diluent.

In a further aspect, compositions are provided comprising one or morechemotherapeutic or radiotherapeutic agent and an oligonucleotide whichis targeted to a mRNA encoding mammalian KSR and which inhibits KSRexpression.

The preparation of therapeutic compositions which contain nucleic acids,oligonucleotides, or analogs as active ingredients is well understood inthe art. Such compositions can be prepared as injectables, either asliquid solutions or suspensions. Solid forms suitable for solution in,or suspension in, liquid prior to injection can also be prepared. Thecompositions may be prepared in solid pill form, including slow releaseformulations. The composition may be in a patch form for transdermalapplication, particularly in slow release format. The preparation canalso be emulsified. The active therapeutic ingredient is often mixedwith excipients which are pharmaceutically acceptable and compatiblewith the active ingredient. Suitable excipients are, for example, water,saline, dextrose, glycerol, ethanol, or the like and combinationsthereof. In addition, if desired, the composition can contain minoramounts of auxiliary substances such as wetting or emulsifying agents,pH buffering agents which enhance the effectiveness of the activeingredient.

A nucleic acid or oligonucleotide can be formulated into the therapeuticcomposition as neutralized pharmaceutically acceptable salt forms.Pharmaceutically acceptable salts include the acid addition salts(formed with the free amino groups of the polypeptide or antibodymolecule) and which are formed with inorganic acids such as, forexample, hydrochloric or phosphoric acids, or such organic acids asacetic, oxalic, tartaric, mandelic, and the like. Salts formed from thefree carboxyl groups can also be derived from inorganic bases such as,for example, sodium, potassium, ammoninum, calcium, or ferrichydroxides, and such organic bases as isopropylamine, trimethylamine,2-ethylamino ethanol, histidine, procaine, and the like.

The therapeutic nucleic acid-, oligonicleotide-, analog- or activefragment-containing compositions may be administered intravenously, asby injection of a unit dose, for example. The term “unit dose” when usedin reference to a therapeutic composition of the present inventionrefers to physically discrete units suitable as unitary dosage forhumans, each unit containing a predetermined quantity of active materialcalculated to produce the desired therapeutic effect in association withthe required diluent; i.e., carrier, or vehicle.

The therapeutic compositions may further include an effective amount ofthe nucleic acid or oligonucleotide, and one or more of the followingactive ingredients or agents: a chemotherapeutic agent, aradiotherapeutic agent, an immunomodulatory agent, an anti-mitoticagent.

The pharmaceutical compositions of the present invention may beadministered in a number of ways depending upon whether local orsystemic treatment is desired and upon the area to be treated.Administration may be topical (including ophthalmic, vaginal, rectal,intranasal, transdermal), oral or parenteral. Parenteral administrationincludes intravenous drip or infusion, subcutaneous, intraperitoneal orintramuscular injection, pulmonary administration, e.g., by inhalationor insufflation, or intrathecal or intraventricular administration. Fororal administration, it has been found that oligonucleotides with atleast one 2′-substituted ribonucleotide are particularly useful becauseof their absortion and distribution characteristics. U.S. Pat. No.5,591,721 (Agrawal et al.) and may be suitable for oral administration.Formulations for topical administration may include transdermal patches,ointments, lotions, creams, gels, drops, suppositories, sprays, liquidsand powders. Conventional pharmaceutical carriers, aqueous, powder oroily bases, thickeners and the like may be necessary or desirable.Coated condoms, gloves and the like may also be useful. Compositions fororal administration include powders or granules, suspensions orsolutions in water or non-aqueous media, capsules, sachets or tablets.Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids orbinders may be desirable. Compositions for parenteral, intrathecal orintraventricular administration may include sterile aqueous solutionswhich may also contain buffers, diluents and other suitable additives.In addition to such pharmaceutical carriers, cationic lipids may beincluded in the formulation to facilitate oligonucleotide uptake. Onesuch composition shown to facilitate uptake is Lipofectin (BRL BethesdaMd.).

Dosing is dependent on severity and responsiveness of the condition tobe treated, with course of treatment lasting from several days toseveral months or until a cure is effected or a diminution of diseasestate is achieved. Optimal dosing schedules can be calculated frommeasurements of drug accumulation in the body. Persons of ordinary skillcan easily determine optimum dosages, dosing methodologies andrepetition rates. Optimum dosages may vary depending on the relativepotency of individual oligonucleotides, and can generally be calculatedbased on IC50s or EC50s in in vitro and in vivo animal studies. Forexample, given the molecular weight of compound (derived fromoligonucleotide sequence and chemical structure) and an effective dosesuch as an IC50, for example (derived experimentally), a dose in mg/kgis routinely calculated.

The oligonucleotides of the invention are also useful for detection anddiagnosis of KSR expression. For example, radiolabeled oligonucleotidescan be prepared by radioactive (e.g. ³²P) labeling at the 5′ end or 3′end (including with polynucleotide kinase), contacted with tissue orcell samples suspected of KSR expression or of gƒ-Ras and unboundoligonucleotide removed. Radioactivity remaining in the sample indicatesbound oligonucleotide (which in turn indicates the presence of KSR or ofgƒ-Ras) and can be quantitated using a scintillation counter or otherroutine means. Radiolabeled oligonucleotide can also be used to performautoradiography of tissues to determine the localization, distributionand quantitation of KSR or gƒ-Ras expression for research, diagnostic ortherapeutic purposes. In addition, the radiolabel may have a therapeuticeffect in promoting cell death or blocking cellular proliferation.Analogous assays for fluorescent detection of raf expression can bedeveloped using oligonucleotides of the invention which are conjugatedwith fluorescein or other fluorescent tag instead of radiolabeling.

The oligonucleotides of the present invention may be labeled with adetectable label. In particular aspects, the label may be selected fromenzymes, chemicals which fluoresce and radioactive elements. In theinstance where a radioactive label, such as the isotopes ³H, ¹⁴C, ³²P,³⁵S, ³⁶Cl, ⁵¹Cr, ⁵⁷Co, ⁵⁸Co, ⁵⁹Fe, ⁹⁰Y, ¹²⁵I, ¹³¹I, and ¹⁸⁶Re knowncurrently available counting procedures may be utilized. In the instancewhere the label is an enzyme, detection may be accomplished by any ofthe presently utilized colorimetric, spectrophotometric,fluorospectrophotometric, amperometric or gasometric techniques known inthe art. A number of fluorescent materials are known and can be utilizedas labels. These include, for example, fluorescein, rhodamine, auramine,Texas Red, AMCA blue and Lucifer Yellow. A particular detecting materialis anti-rabbit antibody prepared in goats and conjugated withfluorescein through an isothiocyanate. Enzyme labels are also useful,and can be detected by any of the presently utilized colorimetric,spectrophotometlic, fluorospectrophotometric, amperometric or gasometrictechniques. The enzyme is conjugated to the selected particle byreaction with bridging molecules such as carbodiimides, diisocyanates,glutaraldehyde and the like. Many enzymes which can be used in theseprocedures are known , including but not limited to peroxidase,β-glucuronidase, β-D-glucosidase, β-D-galactosidase, urease, glucoseoxidase plus peroxidase and alkaline phosphatase. U.S. Pat. Nos.3,654,090; 3,850,752; and 4,016,043 are referred to by way of examplefor their disclosure of alternate labeling material and methods.

The invention includes additional compositions which can inhibit theexpression of a protein, in particular KSR, at the transcriptional levelby blocking translation of KSR mRNA or by facilitating destruction ordestabilization of the RNA such that translation cannot efficiently takeplace. In this aspect, the invention provides a ribozyme that cleavesKSR mRNA.

Ribozymes are RNA molecules possessing the ability to specificallycleave other single stranded RNA molecules in a manner somewhatanalogous to DNA restriction endonucleases. Ribozymes were discoveredfrom the observation that certain mRNAs have the ability to excise theirown introns. By modifying the nucleotide sequence of these RNAs,researchers have been able to engineer molecules that recognize specificnucleotide sequences in an RNA molecule and cleave it (Cech, 1988.).Because they are sequence-specific, only mRNAs with particular sequencesare inactivated.

Investigators have identified two types of ribozymes, Tetrahlymena-typeand “hammerhead”-type. (Hasselhoff and Gerlach, 1988) Tetrahlymena-typeribozymes recognize four-base sequences, while “hammerhead”-typerecognize eleven- to eighteen-base sequences. The longer the recognitionsequence, the more likely it is to occur exclusively in the target mRNAspecies. Therefore, hammerhead-type ribozymes are preferable toTetrahlymena-type ribozymes for inactivating a specific mRNA species,and eighteen base recognition sequences are preferable to shorterrecognition sequences.

The use of RNA interference strategies to inhibit the expression of KSRis further embodied in the invention. Thus, methods of RNA interferenceand small interfering RNA compositions are included in the methods andcompositions of the present invention. RNA interference refers to thesilencing of genes specifically by double stranded RNA (dsRNA) (Fine, A.et al (1998) Nature 391;806-811). In one embodiment, short or smallinterfering RNA (siRNA) is utilized (Elbashir, S.M. et al (2001) Nature411:494-498). In addition, long double stranded RNA hairpins may beemployed (Tavernarakis, N. et al (2000) Nature Genet 24:180-183; Chuang,C. F. and Meyerowitz, E. M. (2000) PNAS USA 97:4985-90; Smith, N A et al(2000) Nature 407:319-20). Virus-mediated RNA interference against K-Rashas been described (B rummelkamp, T.R. et al (2002) Cancer Cell2:243-247).

The invention may be better understood by reference to the followingnon-limiting Examples, which are provided as exemplary of the invention.The following examples are presented in order to more fully illustratethe preferred embodiments of the invention and should in no way beconstrued, however, as limiting the broad scope of the invention.

EXAMPLES

These studies demonstrate that mammalian KSR integrates signalingthrough the EGFR/Ras/MAPK signaling module. That EGFR, Ras and KSR areon the same signaling pathway in mammalian cells is supported by theunusual hair follicle phenotype manifested in EGFR knockout mice andrecapitulated in the KSR knockout, by the attenuation of EGF-inducedMAPK signaling in MEFs, and by the abrogation of EGFR-/Ras-mediatedtumorigenesis in multiple experimental models. Further, genetic andpharmacologic approaches identified KSR as required for various aspectsof tumorigenesis in vitro and in vivo. In vitro, loss of KSR functionreduced proliferation of MEFs, A431 and MCF-7 cells, abrogatedRas-mediated MEF transformation, and attenuated A431 and MCF-7 cellinvasion. In vivo, inactivation of KSR antagonized ν-Ha-Ras-mediatedtumor formation and growth of an established EGFR-driven tumor thatrequires wild type Ras for neoplastic progression. As in C. elegans^(2,3), KSR appears dispensable, for the most part, for normaldevelopment, but required when increased signaling through the EGFR/Raspathway is necessary, as occurs acutely in response to EGF stimulationor chronically in Ras-mediated tumors. This suggests that pharmacologicinactivation might yield a therapeutic gain. Indeed, the resultspresented herein, including in vivo models of Ras-mediatedtumorigenesis, show significant inhibition of cell proliferation andcell invasion on inactivation of ksr. These studies demonstrate the useof ksr antisense oligonucleotides (KSR-AS ODNs) as a therapeuticapproach in cancer and tumorigenesis, particularly K-Ras mediatedtumorigenesis.

Example 1

Abstract

In Drosophila melanogaster and Caeniorhabditis elegans, KinaseSuppressor of Ras (KSR) positively modulates Ras/mitogen-activatedprotein kinase (MAPK) signaling either upstream of or parallel toRaf¹⁻³. The precise signaling mechanism of mammalian KSR, and its rolein Ras-mediated transformation, however, remains uncertain. Utilizingcells markedly overexpressing recombinant KSR, some groups reported KSRinhibits MAPK activation and Ras-induced transformation⁴⁻⁶ while othersobserved enhancing effects⁷⁻¹⁰. Evidence suggests these discrepanciesreflect gene dosage effects¹¹. To gain insight into KSR function invivo, we generated mice homozygous null for KSR. ksr^(−/−) mice areviable and without major developmental defects. Newborn mice, however,display a unique hair follicle phenotype previously observed inEGFR-deficient mice, providing genetic support for the notion that EGFR,Ras and KSR are on the same signaling pathway in mammals. Embryonicfibroblasts from ksr^(−/−) animals were defective in EGF activation ofthe MAPK pathway, and displayed diminished proliferative potential andimpaired Ras-dependent transformability. Tumor formation in Tg.AC mice,resulting from skin-specific ν-Ha-ras expression, was abrogated in theksr^(−/−) background. Thus, evidence presented herein suggests KSRtransduces EGFR-/Ras-mediated neoplasia, which may be potentiallytargeted by anti-KSR therapeutic strategies.

Introduction

Kinase suppressor of Ras (KSR) was identified in Drosophiulamelanogaster and Caenorhabditis elegans as a positive modulator ofRas/mitogen-activated protein kinase (MAPK) signaling either upstream ofor parallel to Raf (1-3). Although an intensive effort has been directedat elucidating the biochemical properties of mammalian KSR, its precisesignaling mechanism remains uncertain. In particular, its role inRas-mediated transformation has not been addressed convincingly. Somegroups reported KSR inhibits MAPK activation and Ras-inducedtransformation (4-6) while others observed enhancing effects (8-10).These experiments utilized cell systems overexpressing recombinant KSRto levels far beyond endogenous KSR, and evidence suggests thesediscrepancies might reflect gene dosage (11). While we and others arguethe necessity of both the kinase and scaffolding functions of KSR forits optimal activation of the Raf-MAPK cascade (26-30), others believethat KSR signals solely via its scaffolding function (9, 12, 31).

To gain insight into the in vivo function of KSR, we generated a mousehomozygous null for KSR. ksr^(−/−) mice are viable and without majordevelopmental defects. Newborn mice, however, display a unique hairfollicle phenotype previously observed in EGFR-deficient mice. Mouseembryonic fibroblasts (MEFs) from ksr^(−/−) animals displayed diminishedproliferative potential and impaired oncogenic ν-Ha-Ras-dependenttransformation. Moreover, EGF and TPA activated the MAPK cascade to asimilar extent in MEFs, yet only c-Raf-1 activation by mitogenic dosesof EGF depended on ksr. The KSR knockout mouse thus allows thedelineation of KSR-dependent and independent mechanisms of c-Raf-1activation. Further, tumor formation in Tg.AC mice resulting fromskin-specific ν-Ha-ras expression, which utilizes MAPK signaling fortransformation (32), was abrogated in the ksr^(−/−) background. Thesedefects in proliferation, transformation and tumor formation suggest KSRtransduces some forms of Ras-mediated neoplasia.

Results and Discussion

To investigate the in vivo function of KSR in mammals, we targeted themouse ksr locus to obtain mice deficient in KSR expression. ksr^(−/−)mice were generated by homologous recombination in embryonic stem (ES)cells using the pF9 targeting vector shown in FIG. 1 a. The targetedregion included the starting methionine (ATG codon at nt 83 in ksr cDNA)and the following 74 amino acids encompassing 85% of the KSR unique CA1domain. Two targeted ES clones (FIG. 1 b) were microinjected intoC57BL/6 blastocysts and both resulted in chimeric mice that transmittedthe mutated ksr allele through to the germline. Crosses of the ksr^(+/−)mice generated progeny with genotypes of the expected Mendelianfrequencies. A PCR-based screening strategy was developed to detect boththe wild-type (wt) and mutated alleles from mouse genomic DNA (FIG. 1c).

As previously reported (12), Northern blot analysis revealed wt KSRtranscripts of 6.4 and 7.4 kb. The smaller transcript was detected byembryonic day 7, while the larger transcript was observed from day 11 on(FIG. 1 d). In the adult, numerous tissues expressed ksr transcriptsincluding heart, spleen, lung, thymus, and brain (FIG. 1 e). Kidneydisplayed little if any ksr mRNA, while the larger transcript wasrestricted to brain. The existence of this larger mRNA was recentlyreported by Morrison and co-workers to represent a splice variant ofmurine KSR1, named B-KSR1 (12). Importantly, ksr^(−/−) mice did notexpress detectable levels of either ksr mRNA in any tissue tested (FIG.1 e). KSR1 and B-KSR1 proteins were also not detected by Western blotanalysis in tissues or in mouse embryo fibroblasts (MEFs) from ksr^(−/−)mice (FIG. 1 f). The lack of KSR was also confirmed by RT-PCR analysiswith primers specific for the 3′-UTR of ksr cDNA (not shown). Our datathus suggest that replacement of the 5′ region of ksr including thestart coding site and most of the CA1 domain successfully abolishedexpression of both murine KSR forms.

KSR knockout mice were viable and fertile, with no major developmentaldefects. No gross histologic abnormalities of the major organs wereapparent in young mice or in adults up to one year of age. Animalweight, behavior and brood size were also unaffected in the KSRknockout. However, histologic examination of the skin of 10-day-oldksr^(−/−) mice revealed noticeably fewer hair follicles, which weredisorganized in dermal location (depth) and orientation (direction), andmanifested asynchronous growth (FIG. 2 a vs. 2 b,c). Further, asignificant proportion displayed a serpentine morphology (FIG. 2 b). Inother follicles, the inner root sheath separated from the hair shaft,resulting in formation of blisters or cysts (FIG. 2 c). Strikingly, thisphenotype closely resembles that found in the skin of EGFR-deficientmice (13) (FIG. 2 d). Grossly, egfr^(−/−) mice display short, wavypelage hair and curly whiskers during the first weeks of age, withpelage and vibrissa hairs becoming progressively sparser and atrophicover time, eventually leading to alopecia (13). Although these grossphenotypes were not seen in ksr^(−/−) mice, increased alopecia andsparse hair growth were observed following treatment with the phorbolester 12-O-tetradecanoylphorbol 13-acetate (TPA) compared to similarlytreated ksr^(+/+) controls (not shown). The manifestation of this uniquehair follicle phenotype by both knockouts supports the contention thatEGFR and KSR might be on the same pathway in mice.

To further elucidate the effect of KSR disruption on activation of theEGFR/MAPK pathway, we generated MEFs from ksr^(+/+) and ksr^(−/−)littermates and evaluated their response to low, mitogenic doses of EGFand TPA, two growth stimuli known to activate the MAPK cascade. After 48hr of serum starvation, MAPK activation in response to various doses ofEGF (0.01-100 ng/ml) or TPA (10 nM-1 uM) was determined by Western blotanalysis using the monoclonal and anti-phospho-p44/42 MAPK(Thr²⁰²/Tyr²⁰⁴) antibody. ksr^(−/−) MEFs displayed a significantreduction in EGF- and TPA-induced MAPK (ERK½) activation at all dosesexamined, while total MAPK content remained largely unchanged (FIG. 3A).For EGF stimulation, inhibition of MAPK activation was manifest at dosesas low as 0.01 ng/ml (not shown), whereas at 100 ng/ml EGF, MAPKactivation was partially restored (FIG. 3A, upper panel, Lane 8).

To examine Raf-1 activation under conditions of MAPK inhibition,endogenous Raf-1 was evenly immunoprecipitated from all MEF lysates (notshown) and activity assayed using kinase-inactive MEK (K97M) as asubstrate. While Raf-1 activity was greatly inhibited (>90%) inksr^(−/−) MEFs in response to mitogenic doses of EGF (FIG. 3B, upperpanel, lanes 4 and 6), no inhibition was observed when stimulated with100 ng/ml EGF (FIG. 3B, upper panel, lane 8). Thus, partial inhibitionof MAPK activation in response to 100 ng/ml EGF in ksr^(−/−) MEFs isindependent of Raf-1 activation, likely resulting from the known MAKscaffolding function of KSR. These results indicate that EGF-stimulatedRaf-1 activation in MEFs is dose-dependent and may occur viaKSR-dependent and independent mechanisms, consistent with our previousfindings (28).

The requirement for KSR for TPA-induced c-Raf-1 activation differed fromthat of mitogenic doses of EGF. In contrast to complete inhibition ofc-Raf-1 activation after stimulation with mitogenic doses of EGF upondeletion of ksr, TPA-induced Raf-1 activation was not altered inksr^(−/−) MEFs (FIG. 3B, lower panel). Thus, the use of the KSR knockoutMEFs allows for the definition of two mechanisms of c-Raf-1 activation,a KSR-dependent mechanism necessary for mitogenic EGF stimulation, and aKSR-independent mechanism used by TPA, and perhaps pharmacologic dosesof EGF. Loss of KSR thuis can impact MAPK activation by two mechanisms,via loss of c-Raf-1 activation as well as the MEK scaffolding functionof KSR.

To examine the biologic consequence of MAPK inhibition on cellproliferation in vivo, a proliferation assay was performed using MEFs inthe exponential phase of cell growth. Consistent with reduction insignaling through the MAPK mitogenic pathway, which providesproliferative signals, a 50% reduction in growth rate in ksr^(−/−) MEFSwas observed (FIG. 3C).

To determine the potential impact of KSR inactivation in Ras-mediatedtransformation, c-Myc and Ha-rasV12 constructs were transduced intoksr^(+/+) and ksr^(−/−) early-passage MEFs using high-titerretroviruses, and the ability to grow as colonies in soft agar wasassessed as described (15). While ksr^(+/+) fibroblasts did not formcolonies in soft-agar, they did so in the presence of Myc and Rasoncogenes (not shown). In contrast, ksr^(−/−) MEFs could not betransformed by Ha-rasV12, even though they were immortalized by c-Myc.Taken together, all these results show that inactivation of KSR bygenetic deletion attenuates signaling through the EGFR/Ras/MAPK pathway.

The participation of oncogenic ras in human cancers is estimated to be30% (33) and approximately 25% of skin lesions in humans involvemutations of the Ha-Ras (25% for squamous cell carcinoma and 28% formelanomas) (34,35). Since ksr^(−/−) mice showed a defect in normaldevelopment of the hair follicle, presumably via impairment of EGFRsignaling, we examined the role of KSR in gain-of-function Ras in theskin. For these studies, we employed Tg.AC mice, which harbor oncogenicν-Ha-ras fused to the ζ-globin promoter (16-18), a standardized modelfor the study of two-stage skin carcinogenesis. The ν-Ha-ras transgeneof Tg.AC mice is transcriptionally silent until induced in latentneoplastic cells (putative stem cells) closely associated with the outerroot sheath cells of the hair follicle (19), a site consistent with ourlocalization of KSR in mouse skin (not shown). Tg.AC mice (in FVB/Nstrain background) were crossed with ksr^(−/−) mice (in a mixedC57BL/6:129sv background). F1 offspring heterozygous for the ksr genewere then interbred to obtain F2 offspring carrying the ν-Ha-rastransgene in the ksr^(+/+) and ksr^(−/−) background. To determine ifdisruption of ksr might influence tumorigenesis in this model, wetopically treated the dorsum of F2 mice twice weekly for 15 weeks withvehicle (acetone), or with 5 μg of TPA. Animals were monitored fordevelopment of skin malignancies for 20 weeks.

Initial control studies using RT-PCR to detect the ν-Ha-ras transgenemRNA showed that loss of KSR function in ksr^(−/−) mice had no impact onTPA-induced expression of the oncogenic ν-Ha-ras transgene in the skin(FIG. 4A). However, 70% of Tg.AC transgenic mice in a ksr^(+/+)background developed papillomas, while only 10% in a ksr^(−/−)background displayed papillomas (FIG. 4B). The average number ofpapillomas in our study was 2-4 per mouse in each group. These studieswith Tg.AC mice demonstrate that genetic inactivation of KSR preventsEGFR-/Ras-mediated skin tumorigenesis.

In summary, these studies demonstrate that mammalian KSR integratessignaling through the EGFR/Ras/MAPK signaling module. That EGFR, Ras andKSR are on the same signaling pathway in mammalian cells is supported bythe unusual hair follicle phenotype manifested in EGFR knockout mice andrecapitulated in the KSR knockout, by the attenuation of EGF-inducedMAPK signaling in MEFs, and by the abrogation of EGFR-/Ras-mediatedtumorigenesis in multiple experimental models (see also Example 2).These studies further demonstrate that Raf-1 activation may occur byKSR-dependent and independent mechanisms. We believe this observationmay help to resolve some of the questions regarding upstream elements ofthe Ras/Raf-1-MAPK module and provides new targets and reagents foradditional investigation. In C. elegans (2,3), KSR appears dispensable,for the most part, for normal development, but required when increasedsignaling through the EGFR/Ras pathway is necessary and for some formsof oncogenic Ras-transduced MAPK-mediated tumorigenesis, as occursacutely in response to EGF stimulation or chronically in Ras-mediatedtumors, indicating that KSR inactivation could yield a therapeutic gain,particularly for selective abrogation of the Ras/MAPK signaling of humantumorigenesis.

Methods

Gene targeting. Mouse ksr genomic DNA clones were isolated by screeninga λFixII phage library prepared from mouse strain 129/sv (Stratagene, LaJolla, Calif.) using the 5′ coding region (nt 1-786) of mouse ksr cDNA(Genbank accession # U43585). The mouse ksr cDNA sequence is provided inFIG. 12A. The targeting vector pF9 was constructed by inserting a 2.5-kbSpel-SmaI fill-in fragment from the 5′ end of the mouse ksr genomicclone into the NotI fill-in site of pPGK-NTK vector (a gift from Dr.Frank Sirotnak). A 6.3-kb SpeI-HindIII fill-in fragment from the 3′downstream region of the mouse ksr genomic clone was inserted into thevector at the ClaI fill-in site. The resulting plasmid was linearizedwith KpnI and electroporated into 129/Sv-derived W9.5 ES cells(Chrysalis DNX Transgenic Sciences, Princeton, N.J.). Two hundredG418/Gancyclovir-resistant ES cell clones were analyzed by Southern blotusing a 0.6 kb BglII-SpeI probe derived from genomic sequences locatedimmediately outside (5′) those present in pF9. This probe hybridizes toa 5.7-kb DNA fragment for the wt ksr allele and a 3.1-kb fragment fromthe disrupted allele. Heterozygous ES cells were microinjected intoblastocyst-stage C57BL/6mouse embryos at the Sloan-Kettering Institute'sTransgenic Core Facility. Injected blastocysts were then transplantedinto the uterus of pseudopregnant C57BL/6 mice. Chimeric males werecrossed to C57BL/6 females. Germline transmission was monitored bySouthern blot in agouti F1 offspring. For mouse genotyping, genomic DNAwas isolated from mouse tails with the DNeasy kit (Qiagen Inc.,Valencia, Calif.) and was either digested with BglII and XhoI andexamined by Southern blot as for ES cells, or analyzed by PCRamplification with two sets of primers. Primers for the wt allele werederived from the cDNA sequence of mouse ksr CA1 domain: upstream primer,5′-TATCTCCATCGGCAGTCT-3′ (SEQ ID NO:20), downstream primer,5′-TCGACGCTCACACT TCAA-3′ (SEQ ID NO:21). The primers for the mutantallele were from the sequence of the neomycin phosphotransferase gene:upstream primer, 5′-CTGACCGCTTCCTCGTG-3′ (SEQ ID NO:22); downstreamprimer, 5′-ATAGAGCCCACCGCATCC-3′(SEQ ID NO:23). The size of the expectedproduct is 493-bp for the wt and 312-bp for the disrupted allele.Standard PCR conditions were employed: initial denaturation of 5 min at94° C., followed by 30 cycles with annealing at 56° C., extension at 72°C., and denaturation at 94° C., all for 30 sec.

Northern and western blot analysis. Poly A⁺RNA was prepared from adultmouse tissues using the Oligotex kit from Qiagen Inc. (Valencia,Calif.). The blots were hybridized with a specific ³²P-labeled probecorresponding to the CA2-CA4 domains of murine ksr cDNA (1.47-kb). Forembryonic tissues, we used a Mouse Embryo MTN Blot (BD Biosciences, SanDiego, Calif.). Protein homogenates were prepared from ksr^(+/+) andksr^(−/−) tissues, or MEFs in RIPA buffer and fractionated by SDS-PAGE(100 μg protein/lane). KSR expression was detected by western blot witha mouse monoclonal anti-KSR antibody (BD Biosciences, San Diego, Calif.)or a goat polyclonal anti-KSR antibody generated to amino acids 855 to871 of KSR (c-19, Santa Cruz Biotechnology, Santa Cruz, Calif.). MEK andMAPK activation in MEFs were detected by western blot withanti-phospho-MEK and anti-phospho-MAPK specific antibodies: polyclonalanti-MEK, polyclonal anti-p44/42 MAPK, monoclonal anti-phospho-p44/42MAPK (Thr²⁰²/Tyr²⁰⁴) and polyclonal anti-phosph-MEK½ (Ser²¹⁷/Ser²²¹)(Cell Signaling, Beverly, Calif.).

Histology. Skin tissues were collected from 10-day old ksr^(+/+),ksr^(−/−) and egfr^(−/−) (kindly provided by Dr. Laura Hansen) mice andfixed for 15-18 hours in 10% neutral buffered formalin, washed 2 hoursin 70% ethanol and embedded in paraffin blocks. The blocks weresectioned 4-6 μm thick, placed on glass slides and stained withhematoxylin and eosin.

MEF studies. MEFs, derived from ksr^(+/+) and ksr^(−/−) day 12-13embryos, were prepared as described¹⁵. 0.25×10⁶ early passage MEFs(PDL<6) were seeded in 6-well plates and grown in DMEM supplemented with10% FBS for 24 h at 37° C. After 48 h in serum-free medium, cells werestimulated with 0.05-1.0 ng/ml EGF for 3 min, washed with PBS and lysedin 0.2 ml of NP-40 lysis buffer (20 mM Tris-HCl, 137 mM NaCl, 2 mM EDTA,10% Glycerol, 1% Nonidet P-40 plus protease and phosphatase inhibitors).Raf-1 activity was performed as previously described (27). Briefly, 300ug of total lysate was immunoprecipitated with a polyclonal anti-Raf-1antibody (Upstate Biotechnology, Lake Placid, N.Y.), washed with NP-40buffer containing 0.5M NaCl and incubated with the kinase-dead GST-MEK-1(K97M). Activated MEK-1 was visualized by Western blot with a polyclonalanti-phospho-MEK antibody (Cell Signaling, Beverly, Calif.). To analyzecell proliferation, 0.15×10⁶ ksr^(+/+) or ksr^(−/−) low-passage MEFswere seeded on 60 mm plates and counted at the indicated time points byhemacytometer. Data (mean+/−SD) are compiled from three independentexperiments. To assess transformation capacity, MEFs from ksr^(+/+) andksr^(−/−) mice were transduced sequentially with retroviral plasmidspWZL-Hygro-c-myc and pBabe-Puro-H-RasV12 (kindly provided by Scott Lowe,Cold Spring Harbor Laboratories), resuspended in 0.3% noble agar andseeded in 60 mm plates as described (15). Colonies consisting of atleast 50 cells were counted after 3 weeks.

Generation of Tg.AC/ksr^(−/−) mice. Homozygous male and female Tg.ACtransgenic mice (16) were obtained at 3-4 week of age from Charles RiverLaboratories Inc. (Wilmington, Mass.). To produce the target population,ksr^(−/−) mice were first bred to hemizygous Tg.AC mice containing theν-Ha-ras transgene. The resulting F1 females and males, heterozygous forksr and hemizygous for the Tg.AC transgene, were then bred to obtainoffspring in the ksr background. Nonresponder Tg.AC mice (17) wereexcluded from the study group. Presence of the Tg.AC transgene wasdetermined by PCR amplification as follows: initial denaturation of 1min 10 sec at 74° C., followed by 30 cycles with annealing at 55° C. forImin, extension at 72° C. for 3 min, and denaturation at 94° C. for 1min. The sequence of the Forward Primer was5′-GGAACCTTACTTCTGTGGTGTGAC-3′ (SEQ ID NO: 13), and the sequence of theReverse Primer was 5′-TAGCAGACACTCTATGCCTGTGTG-3′ (SEQ ID NO: 14). PCRresults were confirmed by Southern blot analysis as described (17).

Skin tumor experiments. Mice were treated twice weekly with 5 μg TPA(Sigma Chemical Company, St. Louis, Mo.) for 15 weeks and observed forpapilloma development as described (16). Offspring from the originalTg.AC mice in the FVB/N background from Charles River Laboratory wereused as controls. Papillomas were counted weekly for 20 weeks. ν-Ha-rastransgene expression in skin after TPA treatment was assessed by nestedPCR as described (24).

References

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Identification of B-KSR1, a novel brain-specific    isoform of KSR1 that functions in neuronal signaling. Mol Cell Biol    20, 5529-39 (2000).-   13. Hansen, L. A. et al. Genetically null mice reveal a central role    for epidermal growth factor receptor in the differentiation of the    hair follicle and normal hair development. Am J Pathol 150, 1959-75.    (1997).-   14. Wennstrom, S. & Downward, J. Role of phosphoinositide 3-kinase    in activation of ras and mitogen-activated protein kinase by    epidermal growth factor. Mol Cell Biol 19, 4279-88 (1999).-   15. Serrano, M., Lin, A. W., McCurrach, M. E., Beach, D. &    Lowe, S. W. Oncogenic ras provokes premature cell senescence    associated with accumulation of p53 and p16INK4a. Cell 88, 593-602    (1997).-   16. Leder, A., Kuo, A., Cardiff, R. D., Sinn, E. & Leder, P.    ν-Ha-ras transgene abrogates the initiation step in mouse skin    tumorigenesis: effects of phorbol esters and retinoic acid. 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Example 2

Abstract

Given the prevalence of oncogenic ras mutations in human cancers,selective inactivation of gain-of-function (gƒ) Ras signaling representsa highly attractive therapeutic approach, although it has not beenachieved clinically. Here, gƒRas signaling was targeted indirectly viagenetic or pharmacologic inactivation of Kinase Suppressor of Ras1(KSR1), an immediate downstream effector selective for gƒRas. KSR1inactivation abrogated gƒRas-mediated tumorigenesis induced byconstitutively activated epidermal growth factor receptor or oncogenicK-Ras mutation in several human tumor cell lines and in nude micexenografts. Inhibition of ksr1 via continuous infusion of KSR antisenseoligonucleotides (AS-ODNs) prevented growth of oncogenic K-ras-dependenthuman PANC-1 pancreatic and A549 non-small-cell lung carcinoma (NSCLC)xenografts in nude mice, effected regression of established PANC-1tumors, and inhibited A549 lung metastases, without apparent toxicity.These studies suggest KSR AS-ODNs as a treatment for gƒRas-dependenthuman malignancies, in particular pancreatic cancer for which there ispresently no effective curative therapy.

Introduction

Adenocarcinoma of the exocrine pancreas represents the fourth-leadingcause of cancer-related mortality in Western countries. Treatment hashad limited success and the five-year survival remains less than 5% witha mean survival of 4 months for patients with surgically unresectabletumors (1,2). While oncogenic activation of H-, K-, and N-Ras arisingfrom single nucleotide substitutions has been observed in 30% of humancancers (5), over 90% of human pancreatic cancer manifest the codon 12K-ras mutation (3-5). This point mutation can be identified early in thecourse of the disease when normal cuboidal pancreatic ductal epitheliumprogresses to a flat hyperplastic lesion, and is considered causative inthe pathogenesis of pancreatic cancer (6,7). The regulation of oncogenicK-ras signaling in human pancreatic cancer, however, remains largelyunknown. While various therapeutic strategies have been developed toinactivate key components of the Ras-Raf-MAPK cascade, specificinhibition of gƒRas action has not been achieved clinically (8,9).

Recent studies demonstrated that Kinase Suppressor of Ras (KSR1)positively modulates the Ras/MAPK signaling arm of the EGFR/Ras pathway.KSR1 acts downstream of Ras, either upstream of or parallel to Raf inDrosophila and C. elegans (10-12). Mammalian forms of KSR1, identifiedon the basis of sequence homology (12), suggest that KSR signaling isevolutionarily conserved. The precise mechanisms of mammalian KSRsignaling and its biological functions, however, remain largely unknown.Genetic studies from ksr1-deficient C. elegans and mice demonstrate thatwhile ksr1 is dispensable for normal development (10,11,13), it may beobligate for gƒRas signaling through the MAPK cascade (see Example 1above). In C. elegans, KSR loss-of-function (lf) reverts thegƒRas-mediated multivulva phenotype (10,11) caused by the same codon 13mutation that confers oncogenic potential onto mammalian Ras.

To elucidate the role of KSR1 in Ras-mediated human malignancies,especially in pancreatic cancer, and to explore the feasibility ofemploying KSR1 as a therapeutic target, antisense approaches wereemployed to genetically and pharmacologically inactivate mammalian ksr1.We report here that both approaches to KSR1 inactivation abrogated gƒRassignaling of tumoligenesis, either via constitutively activated EGFR oroncogenic K-Ras mutation. Further, antisense-mediated inhibition of ksr1gene expression via continuous infusion of KSR AS-ODNs prevented theoncogenic K-ras-dependent growth of human PANC-1 pancreatic and A549non-small-cell lung carcinoma (NSCLC) xenografts in nude mice, elicitedregression of established PANC-1 tumors, and inhibited A549 lungmetastases without apparent toxicity. These studies demonstrate that KSRAS-ODNs might represent a tumor-specific therapeutic agent for thetreatment of oncogenic K-ras-dependent human malignancies.

Results

Inhibition of ksr1 gene expression induces morphologic chances in A431cells.

In C: elegais, KSR1 regulates gƒRas signaling of vulval development, apathway initiated through LET-23, the EGFR homolog (10,11). To explorethe role of mammalian KSR1 in EGFR-mediated tumorigenesis, we employedthe A431 human epidermoid carcinoma tumor line in which tumor growth isdriven through wild type Ras by a 100-fold excess of activated EGFR/HER1(10⁷ receptors/cell) (14). We generated A431 cell lines stablyexpressing inducible forms of wild type KSR1 (KSR-S), antisense KSR1(KSR-AS) and dominant negative KSR1 (DN-KSR) using the Retro-Tet-Offsystem. While Flag-tagged KSR-S and DN-KSR were expressed to similarlevels (FIG. 5A), stable expression of the KSR-AS resulted in a 60%reduction of endogenous KSR expression (FIG. 5B). Further, doxycycline(Dox) treatment elicited a dose-dependent inhibition of KSR-S expression(FIG. 5C), and Dox withdrawal following its addition effectivelyrestored KSR-S expression (not shown). Similar results were found in theDN-KSR and KSR-AS cells (not shown). These observations indicate thatthe KSR-Tet-Off system is tightly regulated by Dox.

The effect of manipulating KSR1 levels on the morphology of stablytransfected A431 cells was examined first. While non-transfected (notshown), vector-transfected and KSR-S-transfected A431 cells displayedthe similar cobblestone morphology of poorly differentiated squamousepithelial cells (FIG. 5D), abrogation of ksr1 expression by KSR-ASproduced a marked change in cell morphology. The KSR-AS cell somatagradually enlarged and flattened, cytoplasmic processes retracted, andcells grew in a more scattered pattern (FIG. 5D). Further, these cellsbecame multinucleated (FIG. 5E), indicative of the failure to completecytokinesis with a resultant proliferation defect (see below) (15).Phase-contrast microscopy reveals that while over 80% of KSR-AS cellscontained multiple nuclei, multinucleated cells were rarely seen (<8%)in control or KSR-S cells. Similar morphologic changes were observed inDN-KSR cells (FIG. 5D and SE) indicating that inhibition of ksr1 geneexpression in A431 cells might have profound effects on EGFR-mediatedbiological events in this tumor cell line.

Inhibition of ksr1 oene expression attenuates A431 tumorigenesis. Toassess the consequences of KSR1 inhibition on the malignant propertiesof A431 cells and their response to EGF in vitro, cell proliferation,invasion and transformation assays were performed. When KSR-S wasoverexpressed by A431 cells, both baseline and EGF-stimulatedproliferation (FIG. 6A), invasion (FIG. 6C) and transformation (FIG. 6D)were markedly enhanced. In contrast, depletion of ksr1 expression inKSR-AS cells resulted a significant inhibition of baselineproliferation, invasion and transformation (FIG. 6, p<0.05 in eachcase), and the abrogation of EGF responses (FIG. 6). The DN-KSR effectwas similar to that observed with KSR-AS (FIG. 6).

Consistent with the observed alterations in cell growth, KSRsignificantly impacted cell cycle distribution as determined by FACSanalysis (FIG. 6B). While there was a significant elevation of S-phasecells in exponentially growing KSR-S cells, a sharp reduction in S-phasecells coupled with a concomitant increase of G2/M-phase cells wasobserved in KSR-AS cells compared to vector-transfected controls (p<0.05in each case). These observations were confirmed by Ki-67 staining (notshown). The specificity of KSR-S and KSR-AS in mediating stimulation andinhibition, respectively, of proliferation, invasion and transformationwas confirmed by turning off the KSR-S and KSR-AS expression by Doxtreatment (not shown). These observations demonstrate that whileoverexpression of KSR enhanced the neoplastic properties of A431 cells,inactivation of KSR by KSR-AS or DN-KSR rendered A431 cells lessmalignant.

To elucidate whether KSR1 down-regulation might have a similaranti-proliferative effect in vivo, 10⁶ KSR-S, KSR-AS, DN-KSR orvector-transfected A431 cells were injected subcutaneously (s.c.) intothe right flank of immunodeficient (nude) mice. While tumor take was100% in mice receiving KSR-S and vector-transfected cells, KSR-S tumorshad an earlier onset (FIG. 7A, left-shifted growth curve, p<0.05), were200% larger in size on day 25, and had 2.5-fold more Ki-67 positivecells than vector-transfected tumors of the comparable size (not shown).Examination of tumor specimens removed on day 25 revealed continuedexpression of Flag-KSR-S (not shown). The specificity of KSR-S inmediating these effects was confirmed by feeding a group of KSR-Stumor-bearing mice with Dox-containing water which shut off tumoralKSR-S expression efficiently (not shown), and almost completelyprevented the growth stimulatory effect of KSR-S on A431 tumors (FIG.7A, KSR-S vs. KSR-S+Dox, p<0.01). In contrast, mice injected with 10⁶A431 KSR-AS or DN-KSR cells failed to develop any tumors when observedup to 120 days (FIG. 7A and not shown). When the inocula size wasincreased to 10×10⁶ and prepared in 50% Matrigel, only 1 out of 20 micein each case developed a late onset (day 42 for KSR-AS and day 36 forDN-KSR) slow growing tumor. Further, squamous differentiation wasevident in both the vector- and KSR-S tumors (FIG. 7B (i) and (ii),black arrows), although KSR-S tumors had less kertohyalin granules and ahigher mitotic index (not shown). In contrast, squamous differentiationwas absent from KSR-AS and DN-KSR tumors (FIG. 7B (iii) and (iv)).Moreover, consistent with our observations in vitro, 25% of the KSR-ASand 18% of the DN-KSR tumor cells were multinucleated in vivo (FIG. 7B(iii) and (iv), black arrows, and (v)).

These observations demonstrate that inhibition of A431 tumorigenesis byKSR-AS involves attenuation of proliferation and induction ofmultinucleation. To confirm that prevention of A431 tumorigenesis byKSR-AS is due to inhibition of ksr1 expression by KSR-AS, we designedphosphorothioate AS-ODNs against the unique CA1 domain (SEQ ID NO: 1)(amino acids 42-82 (SEQ ID NO:2)) of KSR1, which is conserved betweenthe mouse and human (12), to inactivate ksr1 expressionpharmacologically. Among the AS-ODNs tested, the AS-ODN againstnucleotides 214 to 231 (SEQ ID NO:5) of KSR1 (designatedAS-ODN1(214-231)), which has no sequence homology to any other mammaliangene, exhibited the most potent and specific antisense effect, and waschosen for further characterization. In vitro treatment of A431 cellswith 1 μM KSR AS-ODN (SEQ ID NO: 8) for 24 h resulted in a 90% reductionof endogenous KSR1 expression as determined by immunofluorescencestaining (FIG. 8A) and Western blotting (not shown), while control ODNshad no apparent effect (FIG. 8A and not shown). Moreover, expression ofother cellular proteins including the EGFR, H-Ras, c-Raf-1 and MAPK wasnot altered by treatment with KSR AS-ODN or control ODNs (not shown),indicating that the antisense effect was specific for KSR. Similar toinactivation of KSR by stable expression of full-length KSR-AS, KSRAS-ODN treatment attenuated A431 cell proliferation (FIG. 8B) andinvasion (FIG. 8C) in a dose-dependent fashion (p<0.05). At 1 mM, KSRAS-ODN inhibited A431 cell proliferation and invasion by 80% and 70%,respectively. In contrast, Control-ODN (FIG. 8C), which lacks homologyto any mammalian gene (16), or Sense-ODN or mismatch AS-ODNs (notshown), were ineffective.

To assess the antitumor activity of KSR AS-ODNs in vivo, AS-ODNs orcontrol ODNs were delivered via continuous s.c. infusion to providesustained tumor exposure. Infusion was initiated two days prior to tumorimplantation in order to reach a steady state ODN plasma level. 10⁶ A431cells were injected s. c. into nude mice to obtain seed tumors of 400mm³. Approximately 50 mg of the freshly prepared seed tumor fragmentswere then transplanted to AS-ODN- or Control ODN-treated mice. Treatmentwith KSR AS-ODN at a low dose of 5 mg/kg/day effectively reduced tumoralKSR1 levels by 85% and attenuated A431 tumor growth by 80% (FIG. 8D,p<0.01), without apparent toxicity, consistent with the known lack oftoxicity of this therapeutic approach (17). In contrast, no antitumoreffects were observed following treatment with vehicle alone (saline) orwith identical doses of the Control-ODN, or Sense-ODN (FIG. 8D and notshown). Similar results were obtained when treatment was initiated usingmice with established A431 tumors of 150 mm³ (not shown). Collectively,these results demonstrate that KSR1 is obligate for EGFR signaling ofA431 tumorigenesis in vivo via hyperactivated wild type Ras. Further,the antitumor activity of KSR AS-ODNs appeared to be achieved viaselective inhibition of ksr1 gene expression with high specificity.These studies suggest that it might be feasible to use KSR AS-ODN toabrogate EGFR/Ras signaling of human tumorigenesis.

Inhibition of ksr1 expression abrogates oncogenic K-ras-mediated humanpancreatic tumorigenesis via specific attenuation of Ras/Raf-MAPKsignaling. To elucidate the importance of KSR1 in mediating oncogenicRas signaling of human tumorigenesis and to explore the therapeuticpotential of KSR AS-ODNs, we employed the human pancreatic cancer PANC-1xenograft mouse model. This tumor manifests the oncogenic codon12-mutation of K-ras. Similar to A431 cells, treatment of PANC-1 cellsin vitro with KSR AS-ODNs attenuated cell proliferation (FIG. 9A),invasion (FIG. 9C) and transformation (FIG. 9D) in a dose-dependentfashion (FIG. 9 and not shown) (p<0.05 in each case). Further, treatmentwith 5 μM AS-ODN led to a 90% reduction of endogenous KSR1 expression(FIG. 9E). To confirm the effectiveness of KSR AS-ODN in inhibitingoncogenic K-ras function, a panel of codon-12 K-ras mutated humanpancreatic cancer cell lines were treated with 5 μM of KSR AS-ODN andassayed for cell proliferation. While cell growth was inhibited by 50 to80% in all cell lines after AS-ODN treatment (p<0.01 each), Sense-ODNhad no apparent effect (FIG. 9B).

We previously demonstrated that KSR1 activation is required for c-Raf-1and subsequent MAPK activation in vitro in response to mitogenic dosesof EGF stimulation (18,19). To molecularly order KSR1 and Raf-1 inoncogenic K-ras signaling, PANC-1 cells were treated with 5 μM AS-ODN,transfected with the dominant positive BXB-Raf-1 and assayed for cellinvasion and transformation. If Raf-1 is downstream of KSR, gƒRaf-1(BXB-Raf-1) should reverse the inhibitory effect of KSR inactivation byAS-ODNs on PANC-1 cell invasion and transformation. Indeed, whileBXB-Raf-1 had no effect on endogenous KSR1 expression (FIG. 9E), itcompletely reversed the inhibitory effect of AS-ODN on PANC-1 cellinvasion (FIG. 9C) and transformation (FIG. 9D). These observationsindicate that c-Raf-1 is epistatic to KSR1, consistent with our in vitrofindings and with the current literature (19-22). Additional studieswere performed to examine the mechanism by which KSR1 inactivationaffected oncogenic Ras-mediated intracellular signaling. For thesestudies, AS-ODN-treated and BXB-Raf-1-transfected PANC-1 cells wereserum-depleted for 48 h and stimulated with 1 ng/ml of EGF. MAPK andPI-3 kinase activation were assayed by Western blot analysis usingphospho-MAPK and phospho-Akt specific antibodies. While AS-ODN treatmentblocked EGF-induced MAPK activation (FIG. 9F, upper panel, lane 6 vs.lane 2), it had no apparent effect on Akt activation (FIG. 9F, lowerpanel, lane 6 vs. lane 2). Sense-ODN had no effect on either MAPK or Aktactivation (FIG. 9F). Moreover, the inhibitory effect of AS-ODN on MAPKactivation could be completely reversed by expression of BXB-Raf-1 (FIG.9F, upper panel, lane 4 vs. lane 2). Total MAPK and Akt content werelargely unaffected by treatment with ODNs or transfection with BXB-Raf-1(FIG. 9F and not shown). These results suggest that abrogation ofoncogenic K-Ras signaling in pancreatic cancer cells by KSR AS-ODN islikely achieved by specific inhibition of the Ras-Raf-MAPK cascade. Totest the therapeutic potential of KSR AS-ODNs to treat human pancreaticcancer, PANC-1 xenografts either derived from 10⁶ cultured PANC-1 cells(FIG. 10A (i)), or from freshly harvested seed PANC-1 tumors (preparedas described above) (FIG. 10A (ii)), were transplanted into nude mice.The steady state plasma AS-ODN levels for the 5 and 10 mg/kg/day dosesof infusion were determined by OliGreen and HPLC assays to be 63 and 123ng/ml, respectively, consistent with that reported in the literatureusing similar doses (23). For PANC-1 tumors arising from the injectedcells, tumors were allowed to reach 100 mm³ prior to the initiation ofAS-ODN treatment. Infusion of AS-ODNs at 10 mg/kg/day for 14 daysresulted in 40% reduction in tumor volume with a 100% response rate(FIG. 10A (i), p<0.05 vs. Control-ODN). A group of AS-ODN treated tumorsthat had regressed were monitored for tumor re-growth after thetreatment was discontinued. Only 1 of 5 tumors exhibited re-growth whilethe rest remained regressed and stable for up to 4 weeks (not shown).For PANC-1 xenografts propagated via serial passage in vivo, continuousinfusion of KSR AS-ODNs, initiated 2 days prior to tumortransplantation, attenuated the growth of PANC-1 tumors in adose-dependent fashion (FIG. 10A (ii)). No apparent toxicity (weightloss, behavioral alteration, organomegaly, inflammation, bleeding) wasobserved at any dose and was confirmed by histologic examination ofnumerous tissues at autopsy (not shown). At 75 mg/kg/day, PANC-1 tumorgrowth was completely abolished and all mice remain tumor-free up to 4weeks after the treatment was discontinued (FIG. 10A (ii) and notshown). In contrast, treatment with vehicle alone (saline), Control-ODN,or Sense-ODN exhibited no antitumor effects at all doses examined (FIG.10A and not shown). These observations support the conclusion that theantitumor effects observed for KSR AS-ODNs occur through an antisensemechanism of action. Similar anti-neoplastic effects of KSR AS-ODN wereobserved in PANC-1 tumors transplanted orthotopically under thepancreatic capsular tissue (not shown).

To confirm the specificity of KSR in mediating K-ras signaling ofpancreatic tumorigenesis, we examined endogenous ksr1 gene expression insaline-, Sense-ODN and AS-ODN-infused PANC-1 tumors. KSR1 expression wasinhibited by 90% in all AS-ODN-treated animals examined, while it waslargely unchanged by saline or Sense-ODN infusion (FIG. 10B), confirminga sequence-specific target effect. As an additional control, the effectof AS-ODN treatment on Ras activation in vivo was measured bydetermining the amount of GTP-Ras in PANC-1 tumors using theGST-RBD-Raf-1 pull down assay as described in Methods. Consistent withthe data of A431 and PANC-1 cells in culture (not shown), AS-ODNtreatment had no apparent effect on Ras activation (FIG. 10C),indicating that signaling events upstream of Ras activation were intactand inactivation of oncogenic K-ras signaling in PANC-1 cells by KSRdepletion occurs downstream of Ras.

To confirm the effectiveness of KSR AS-ODNs in hindering other oncogenicK-ras-dependent human tumors, the codon 12 K-ras mutated A549 humannon-small-cell lung carcinoma model (NSCLC) was selected. For thesestudies, 50 mg A549 seed tumor fragments, prepared similarly to A431 andPANC-1 seed tumors as above, were transplanted s. c. into nude mice.Treatment with KSR AS-ODN was initiated when A549 tumors reached 150mm³. At 10 mg/kg/day, KSR AS-ODN completely inhibited the growth of theestablished A549 tumors while Saline, Control-ODN or Sense-ODN had noapparent effect (FIG. 10D (i)). When animals were sacrificed at the endof the experiment, lungs were resected from Sense-ODN- or AS-ODN-treatedmice and stained with Indian ink to visualize surface lung metastasesderived via systemic dissemination. Control-ODN-treated lungs had anaverage of 8-11 metastatic foci. AS-ODN treatment elicited adose-dependent inhibition of A549 lung metastasis (FIG. 10D (ii),p<0.05). These observations suggest that while KSR1 is obligate forK-ras-dependent primary tumor growth, it may also play an essential rolein the metastatic progression of these tumors. Further, KSR AS-ODN couldbe an effective agent in the management of K-ras-dependent humanmalignancies.

Discussion

The present studies provide evidence that KSR1 is obligate for gƒRassignaling at he tissue level, and that inhibition of ksr1 expressionleads to selective regression of gƒRas-dependent tumors. Previousclinical studies designed to treat gƒRas-dependent tumors by inhibitionof elements of the Ras/Raf-1/MAPK signaling cascade have to date beenlargely unsuccessful (9). While toxicity for most agents has beenacceptable, success of treatment has been limited by lack of specificityin inhibiting different Ras isoforms, which recent data suggest may havedistinct biologic functions (24-26) and lack of selectivity towardsgƒversus physiologic Ras signaling (8,9). Similar problems exists forexperimental drugs designed to inhibit elements of the Raf-1/MEK1/MAPKcascade (9). The present studies on the effects of KSR AS-ODNs providean approach to specifically attenuate gƒRas signaling in the treatmentof Ras-dependent human tumors.

While the targeting of DNA sequences with AS-ODN technology representsan attractive therapeutic approach to the treatment of cancer (27), aprinciple problem with this approach has been the designation ofspecificity of the AS-ODN effect for the gene of interest. We provide anumber of different lines of evidence to support the notion that theinhibition of tumorigenesis observed with KSR AS-ODNs is due toselective inactivation of gƒK-ras signaling via inhibition of ksr1. Ourdata show that genetic inhibition of ksr1 expression by the KSR-ASTet-Off construct yielded comparable antisense-mediated effects in vitroand in vivo as KSR AS-ODNs. Further, various ODNs, designed to controlfor sequence-dependent and sequence-independent non-antisense artifacts,had no effects on ksr1 gene expression, or on tumor growth in cellculture or in vivo. In addition to ODN sequence specificity, the effectsof KSR AS-ODN on ksr1 expression were specific for the intended targetas expression of other genes of the EGFR-Ras-MAPK pathway wasunaffected. Finally, conditional overexpression of KSR-S by A431 cellsdelivered a phenotype opposite to KSR inactivation by KSR-AS, and bothKSR-S and KSR-AS effects were reversible in the Tet-Off system byturning off expression by Dox treatment. These results collectivelyattest that the antitumor effects of KSR AS-ODN are achieved by anantisense mechanism.

The lack of normal tissue toxicity in animals treated with KSR AS-ODNsis consistent with recent reports that ksr1 is dispensable for normaldevelopment in C. elegans and mice (10,11,13, and see Example 1 above).As recent investigations have uncovered a second ksr allele, ksr2 in C.elegans (28) and in mice (29), the lack of tissue toxicity afterdepletion of ksr1 by KSR AS-ODN might be due to compensation by ksr2 fornormal cellular functions. Alternately, the lack of toxicity may reflecttopological distribution of KSR. Recent evidence suggests that elementsof the Ras/Raf-1/MAPK pathway are compartmentalized in more than onetype of membrane microdomains (26,30), and that compartmentalization isassociated with regulation of activity. In this regard, Ras and c-Raf-1associate with sphingolipid enriched microdomains (also known as rafts)in the plasma membrane and with the bulk membrane fraction. Raftassociation, at least for c-Raf-1, appears to involve binding to thesphingolipid ceramide (31). Further, depending on activation status, Rasforms may traffic between compartments (32), with gƒRas preferentiallytargeting the bulk membrane.

Whether KSR, which some groups argue is ceramide-activated (20,33),plays a specific role in the gƒRas activation process, and hence itsinactivation would marginally affect normal cellular function, will bethe topic of future investigation. Lastly, the apparent lack of toxicityof our KSR ODN is not surprising as the phosphorothioate class ofAS-ODNs, the most commonly used AS-ODNs, are generally well tolerated.Administration via continuous infusion in preclinical models and inhuman clinical trials have established that sequence-independenttoxicities (activation of the complement system, prolongation ofactivated partial thromboplastin time and alterations of hematologicalparameters) are usually not encountered at doses at which pharmacologicantisense effects are achieved (34-36).

These studies also suggest that the therapeutic benefit of KSR AS-ODNsmay not be limited to oncogenic K-ras-dependent human cancers, but mightinclude a broader spectrum of tumor types, as our studies with KSRAS-ODNs were found effective against the tumor line A431, which isdriven by hyperactivated wild type Ras. The therapeutic action of KSRAS-ODNs on established tumors in vivo likely involves both inhibition oftumor cell proliferation and induction of tumoral cell death. Theanti-proliferative effect of KSR1 inactivation was evident by a decreasein cells in S phase and the induction of multinuclei phenotype in vitroand in vivo. Additionally, AS-ODN-treated A431 and PANC-1 tumorscontained large necrotic areas (60-80% of the surface of the cutsection). The mechanism of the latter effect, however, remains unknown.Previous studies demonstrated that significant microvascular endothelialapoptosis might also contribute to the anti-tumor effect as a result ofras inactivation (37). However, in our models, only sporadic endothelialcell apoptosis was detected by CD34 and TUNEL staining in PANC-1 tumorstreated with KSR AS-ODNs (not shown). The role of KSR in angiogenesismust await further investigation in more relevant models of tumorangiogenesis.

Another important finding emerging from the present study is that KSRappears required for oncogenic Ras-mediated tumor metastaticprogression. Inhibition of A549 lung metastases with KSR AS-ODNtreatment is in agreement with our preliminary data that MMP-2 and 9activities were increased in A431-KSR-S cells and inhibited inA431-KSR-AS cells (data not shown). Investigations are underway toelucidate the role of KSR in tumor progression.

The effective use of KSR AS-ODNs also provides the potential forimproved understanding of the regulation of critical downstream eventsinvolved in gƒRas signaling, which at the present time, are onlypartially known. Raf-MAPK and PI-3 kinase modules are two establisheddownstream pathways mediating gƒRas signaling of tumorigenesis (38-41).Here we provided evidence that KSR1 functions as a critical mediator ofgƒRas likely via specific regulation of the Raf-1-MAPK signaling arm.Support for this notion is derived from recent studies demonstratingthat MMTV-MT-dependent mammary tumor genesis, signaled primarily via srcand PI-3 kinase via wild type Ras, was not affected in ksr −/− mice(13). In contrast, tumor genesis of oncogenic ν-Ha-Ras-mediatedepidermal skin tumors, signaled through the c-Raf-1/MAPK cascade, wasabrogated in ksr1 −/− mice (Lozano and Kolesnick, unpublished). Further,the present studies with KSR antisense support the molecular ordering ofc-Raf-1 as epistatic to mammalian KSR1, which is consistent with geneticresults from Drosophila and C. elegans (10,11). We believe theseobservations may help to resolve some of the disputes regarding upstreamelements of the Ras/Raf-1/MAPK module.

In summary, the current study provides original observations supportingKSR1 as a new molecular target for the treatment of human malignanciesdependent on gƒRas signaling.

Methods

Cell culture and generation of Retro-Tet-Off A431 cell lines. Humanepidermal carcinoma cell line A431, lung carcinoma cell line A549 andpancreatic cell lines PANC-1, Capan-2, PL-45, HPAF-II, AsPc-1 andMiapaPa-2 were obtained from ATCC (Manassas, Va.). The full-length wildtype mouse ksr1 cDNA, which is over 90% identical to human ksr1 (12),was cloned in both sense (KSR-S) and antisense (KSR-AS) orientationsinto the pRetor-TRE under a doxycycline-inducible promoter inpRetro-Tet-Off (Clontech, Palo Alto, Calif.). DN-KSR (D683A/D700A/R589M)was sub-cloned similarly. A431 cells were infected with medium collectedfrom PT67 packaging cells transfected with KSR-S, KSR-AS, DN-KSR or theempty vector, and maintained under double selection (0.1 mg/ml neomycinand 0.1 mg/ml Hygromycin).

Western blot, immunofluorescence and immunohistochemistry. Total celllysates and tumor lysates were prepared in NP-40 buffer as described(18,42). Immunoprecipitation (IP) or Western blotting (WB) was performedaccording to the manufacturer's protocols with the following antibodies:monoclonal anti-Flag M2 antibody from Sigma (St Louis, Mo.), polyclonalanti-p44/42 MAPK, monoclonal anti-phospho-p44/42 MAPK (Thr202/Tyr204),polyclonal anti-phospho-MEK½ (Ser217/Ser221) and polyclonalanti-phospho-Akt (Ser 473) antibodies from Cell Signaling (Beverly,Calif.), and polyclonal anti-c-Raf-1 antibody from Upstate BiotechnologyInc. (Lake Placid, N.Y.). Endogenous KSR1 expression was determined byimmunoprecipitation and WB analysis from 1 mg of total lysates or byimmunofluorescence microscopy, using the monoclonal anti-KSR antibody(BD Biosciences, San Diego, Calif.) (1:100 dilution) and HRP- orTexas-Red-conjugated goat anti-mouse secondary antibodies, respectively(Molecular Probes, Eugene, Oreg.). Histology and immunohistochemistrywere performed on formalin-fixed, paraffin-embedded tumor or tissuespecimens. 5 mm-cut sections were deparaffinized, rehydrated in gradedalcohols, and H & E stained or immunostained using the avidin-biotinimmunoperoxidase (Vector Laboratories, Burlingame, Calif.) method (43).The following primary antibodies were used: rat anti-mouse CD34 (1:50)antibody from PharMingen (San Diego, Calif.) and polyclonal anti-humanKi67 antibody (1:100) (Vector Laboratories). Diaminobenzidine was usedas the chromogen and hematoxylin as the nuclear counterstain asdescribed (43). Apoptosis were assessed by terminal deoxytransferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL)(Roch, Mannheim, Germany) as described (43).

Proliferation, Matrioel invasion and soft agar transformation assays.2×10⁴ A431 cells or 1-3×10⁴ human pancreatic cells were plated in 6-wellplates. The total number of cells/well was counted at the indicated timepoints to construct cell growth curves. For EGF treatment, 1.0 ng/ml ofEGF was added to the culture and replaced every other day. The invasionassay was performed as described (42). Cells on the underside of thefilters were counted in 10 randomly chosen fields (40× magnification)and reported as an average number of cells invaded per field. For EGFtreatment, cells were replaced with serum-free medium for 2 h prior tothe experiment. The Soft Agar assay was set up in 35 mm culture platescoated with 1.5 ml culture medium containing 0.5% agar and 5% FBS. 5×10³cells were suspended in 1.5 ml medium containing 0.1% agar and 5% FBS,and added to the agar pre-coated plates. Colonies consisting of morethan 50 cells were scored after 14-21 days of incubation using adissecting microscope. For EGF treatment, FBS was omitted from theculture medium.

Cell cycle analysis. Cell cycle distribution was determined by FACSanalysis. For these studies, cell pellets collected from exponentiallygrowing monolayers were washed twice with PBS containing 0.5% FBS andfixed with 100% ethanol for 15 min. Fixed cells were treated withRNase.A (0.1 mg/ml) for 30 min at 37° C. and stained with propidiumiodide (0.05 mg/ml). The proportion of cells in the different phases ofthe cell cycle was calculated from the experimental fluorescencehistograms.

In vitro treatment with KSR AS-ODN. KSR1 AS-ODN(5′-CTTTGCCTCTAGGGTCCG-3′) (SEQ ID NO: 8)(AS-ODN1(214-231)) and KSRsense-ODN (5′-CGGACCCTAGAGGCAAAG-3′) (SEQ ID NO: 15) were generated asphosphorothioate derivatives against nucleotides 214 to 231 (SEQ IDNO: 1) of the unique CA1 domain (amino acids (AAs) 42-82) of KSR1 byGenelink Inc. (Hawthorne, N.Y.). Control ODN(5′-CACGTCACGCGCGCACTATT-3′) (SEQ ID NO: 16) was prepared similarly. Forin vitro studies, ODNs were dissolved in sterile water and delivered tocells by Oligofectamine (Invitrogen, Carlsbad, Calif.) when cells were30-40% confluent according to manufacturer's instructions. Cellproliferation was assayed at the indicated time points. 48 h aftertreatment, invasion and transformation assays were set up as above. Forsome studies, Control- and AS-ODN-treated PANC-1 cells were transfectedwith the dominant positive RSV-Raf-BXB (kindly provided by Dr. JosephBruder, NCI).

Tumor induction and in vivo treatment with KSR AS-ODN. For tumorinduction, 10⁶ cultured tumor cells suspended in 0.1 ml of PBS, or 50 mgof tumor fragments freshly harvested from serial passaged seed tumors,were transplanted subcutaneously into the right lateral flank of 6-8 wkold male athymic NCRnu (Germantown, N.Y.). Tumor growth was measuredevery other day by calipers and tumor volume calculated as described(42). To determine the specificity of KSR-S on A431 tumorigenesis, agroup of KSR-S tumor-bearing mice were fed with Dox-containing water(100 mg/ml). To determine the antitumor activity of KSR-AS ODN in vivo,infusion with Sense-, Control- or AS-ODNs via Alzet osmotic minipumpswas initiated either 2 days prior to tumor transplantation or when tumorreached 100-150 mm³. A 5.0-75 mg/kg body weight/day dose range of ODNwas chosen based on similar AS studies in vivo (34,44)

Ras activation assay. Ras activation status (GTP-Ras) in control ODNs orAS-ODN-treated PANC-1 cells or tumors was measured using the Rasactivation assay kit (Upstate Biotechnology Inc., Lake Placid, N.Y.)according to manufacturer's instructions as described (45).

Statistical analysis. All data were evaluated by the Student's t test(two-tailed) with p<0.05 considered significant.

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Example 3

Additional antisense oligonucleotides were synthesized and tested byproliferation assay in A431 cells as indicated in TABLE 1. Antisensenucleotides and their sequences were selected on the basis of ciiteria(based on hybridization conditions) to have no stable homodimerformation, no hairpin loop formation, no self-complementary sequences,and to have no stable duplex formation (<−6 kcal/mol. Sequences wereselected using the Oligo 4 program (Molecular Biology Insights, Inc.,Cascade, Colo.) and subsequently verified with the Oligo Tech program(Oligo Therapeutics Inc., Wilsonville, Oreg.). Antisenseoligonucleotides were generated against nucleotides 151 to 168(AS-ODN3(151-168) (AS oligo sequence 5′-CAGCCCGCGCAGACTGCC-3′) (SEQ IDNO: 6) and nucleotides 181 to 198 (AS-ODN2(181-198)) (AS oligo sequence5′-GAGGTCGTTAGACACTGA-3′) (SEQ ID NO: 7) of the KSR CA1 domain (bothwere P-S oligonucleotides). These oligonucleotides were tested alongwith the AS-ODN oligonucleotide against nucleotides 214 to 231(AS-ODN1(214-231)) (AS oligo sequence SEQ ID NO: 8) described in Example2. These antisense oligonucleotides represent reverse complements ofnucleic acids encoding the amino acid sequences GSLRGL (SEQ ID NO: 17),AVSNDL (SEQ ID NO: 18) and RTLEAK (SEQ ID NO: 19) of the CA1 domain ofKSR, respectively. A431 cells were transfected with the indicated amountof KSR AS-ODNs and cell proliferation was assessed 72 hr after thetreatment. The effect of AS-ODN on A431 cell proliferation was presentedas percent of inhibition of vehicle-treated (Oligofectamine alone)controls. This is a representation of one of four similar studies. TABLE1 Screening of KSR AS-ODNs by proliferation assay in A431 cells Concen-AS181-198 Control tration of AS151-168 (% AS214-231 ODN (% ODN (nM) (%inhibition) inhibition) (% inhibition) inhibition) 10 0 0 8 2 50 0 5 203 100 3 15 25 2 200 16 23 42 0 400 24 42 67 4 800 38 58 87 3

Example 4

To stablish the specificity of KSR1 in mediating gƒRas signaling, thewell-characterized human chronic myeloid leukemia cell line K562 wasemployed. K562 is Ber-abl-driven and is therefore independent of gƒRassignaling. The specific and dose-dependent inhibition of PANC-1 cellproliferation by AS-ODN treatment is depicted in FIG. 13. PANC-1 andK562 cells were treated with the indicated doses of Control- or AS-ODNsand cell proliferation assays were performed (FIG. 13A). AS-ODN-1 andAS-ODN-2 correspond to nucleotides 214-231 and 181-198 of ksr1 cDNA,respectively. While PANC-1 cell proliferation was inhibited as expected,K562 cell proliferation was unaffected by ODN treatment. Treatment ofK562 cells with 5 μM KSR AS-ODN-1 elicited comparable reduction ofendogenous KSR1 gene expression (over 80%) to that observed in PANC-1cells, as determined by Western blot analysis (FIG. 13B). Nonetheless,inhibition of proliferation was observed only in PANC-1 cells (FIG.13A). Equal loading of the gels was confirmed using total P44/42 MAPK.Note that purified Flag-KSR, which served as a positive control for theWestern blot, migrates slightly slower than endogenous KSR due to theFlag-tag (FIG. 13B). These results provide evidence that gƒRas signalingis specifically coupled to KSR1.

Example 5

The full length mRNA sequence of human KSR1 has been determined. Thesequence is depicted in FIG. 14 (SEQ ID NO: 24). Antisenseoligonucleotides, including those described above, were designed againstthe CA1 domain of human KSR nucleic acid. The human KSR antisenseoligonucleotides are depicted on the annotated human KSR sequence inFIG. 15. Oligonucleotide AS-ODN1(187-204) (5′CTTTGCCTCTAGGGTCCG 3′) (SEQID NO:) against nucleotides 187 to 204 of the human sequence correspondsin sequence to AS-ODN1(214-231) (SEQ ID NO:8) described above. Thus,AS-ODN1 is complementary to the nucleotides at positions 187-204 and214-231 of the human and mouse cDNA, respectively. OligonucleotideAS-ODN3(124-141) (5′CAGCCCGCGCAGACTGCC 3′) (SEQ ID NO:) againstnucleotides 124 to 141 of the human sequence corresponds in sequence toAS-ODN1(151-168) (SEQ ID NO: 6) described above. Thus, AS-ODN3 iscomplementary to the nucleotides at positions 124-141 and 151-168 of thehuman and mouse cDNA sequences, respectively. OligonucleotideAS-ODN2(154-171) is designed against nucleotides 154 to 171 of the humansequence. AS-ODN2 is complementary to nucleotides at positions 154-171and 181-198 of the human and mouse cDNA, respectively. The humansequence differs by a single base pair in the most 5′ bp of theantisense sequence from the mouse sequence in the correspondingposition, with the human AS-ODN2(154-171) sequence being5′GAGGTCGTTAGACACTGC 3′ (SEQ ID NO:) and the mouse sequence being5′GAGGTCGTTAGACACTGA 3′ (SEQ ID NO: 7) (the nucleotide difference is setout in bold). FIG. 16 depicts the annotated mouse KSR cDNA sequence withantisense oligonucleotides indicated. We have compared the originalAS-ODN2(181-198) to the revised human AS-ODN2(154-171) and found theyinhibited proliferation of PANC-1 cells, which are oncogenicK-ras-dependent human pancreatic cells, nearly identically (FIG. 18).

Example 6

We have designed additional potential AS-ODNs against other nucleotidesof human KSR1. These ODNs (AS-ODN4 to AS-ODN12) are marked and annotatedon human KSR1 nucleotide sequences in FIG. 17. TABLE 2 is a list ofthese newly designed ODNs with colTesponding human nucleotide targetsequence. TABLE 2 AS-ODN ID # Target Sequence (nt) (5′-3′) Sequence (5′to 3′) AS-ODN4 ATGGGAGAGAAGGAGGGC  (1-18) GCCCTCCTTCTCTCCCAT AS-ODN5CTGGTCCGTTACATTTGT (205-222) ACAAATGTAACGGACCAG AS-ODN6GTGGCTCCCGGTGAGAGG (247-264) CCTCTCACCGGGAGCCAC AS-ODN7GACTGGCTGTACACTTTC (298-315) GAAAGTGTACAGCCAGTC AS-ODN8GAGGCCGGAGGTGGTGCA (321-338) TGCACCACCTCCGGCCTC AS-ODN9AGATCCCCCGAGACCTCA (351-368) TGAGGTCTCGGGGGATCT AS-ODN10ATGAATGAGGCCAAGGTG (379-396) CACCTTGGCCTCATTCAT AS-ODN11AGTTGGAGTTCATTGGAT (511-528) ATCCAATGAACTCCAACT AS-ODN12GCGGCGGGAAAGTGGCTC (531-548) GAGCCACTTTCCCGCCGC

Example 7 KSR Antisense Oligonucleotides as Radiosensitizers

Background

Although ionizing radiation (IR) remains a primary treatment for humancancers, the failure to respond to radiation therapy limits the efficacyof this modality. Accumulating evidence supports the contention that asignal transduction pathway, analogous to that for cell growth anddifferentiation leads to resistance to IR. More specifically,constitutive activation of the EGFR/Ras pathway via gƒRas orhyperactivation of EGFRs lead to radioresistance in human tumor cells(1-27). The precise mechanisms underlying this action of EGFR and Rasare not well understood. Hence, identification of downstream elements ofEGFR and Ras effector signaling in response to IR is critical to thedevelopment of mechanism-based therapeutic strategies for IR treatment.Of the EGFR and Ras effector signaling pathways, the Ras-Raf-MAPK (10,28-32) and Ras-PI3-Kinase (3,33,34) pathways have been implicated tomediate EGFR/Ras regulation of radiosensitivity to IR. The relativecontribution of these two signaling pathways may be tumor-type specific.

Potential mechanisms underlying EGFR/Ras-mediated radioresistanceinclude: increased capacity for DNA damage repair, acceleratedrepopulation of surviving tumor cells, resistance to IR-inducedapoptosis and lack of IR-induced cell cycle check point delays.IR-induced EGFR/Ras activation results in a pronounced dose-dependentproliferative response via the MAPK signaling cascade, contributing atleast in part, to the mechanism of accelerated proliferation, a causecontributing to the failure of radiotherapy (49,50). As an obligatemediator of the gƒEGFR/Ras-Raf-1-MAPK signaling of tumor cellproliferation (51), inactivation of KSR may increase the efficacy of IRtherapy by abrogating the accelerated repopulation of surviving tumorcells.

In addition to potentially mediating radiation resistance through EGFRand Ras, KSR may also transmit signals for the lipid second messengerceramide. Emerging data suggest that IR acts directly on the plasmamembrane of several cell types, activating acid sphingomyelinase, whichgenerates ceramide by enzymatic hydrolysis of sphingomyelin. Ceramidethen acts as a second messenger in initiating an apoptotic response thatis cell-type and cell-context specific (13, 15, 35-38). In addition tothe tyrosine kinase based mechanism that the EGFR utilizes for KSRactivation, ceramide may also directly activate KSR. In this regard, KSRwas originally identified by Kolesnick and co-workers as ceramideactivate protein kinase (CAPK) (39). While ceramide can signal apoptosisin some cell types through the c-Jun kinase and transcriptionalregulation of gene products, such as Fas ligand or TNFα that mediate thedeath response (13, 14,45), in other cells that contain thepro-apoptotic Bcl-2 family member BAD, ceramide induces apoptosisdirectly through a mechanism involving sequential Ras-dependentactivation of KSR, c-Raf-1, and MEK1 (44, 46-48). In this case, theavailability of a single target, such as BAD (44), converts the MAPKcascade, which is usually proliferative and/or anti-apoptotic, into apro-apoptotic signaling pathway. Therefore, KSR may modulateradiosensitivity by regulation of ceramide-dependent and independentapoptotic responses in a cell-type specific manner. In support of thenotion that KSR may prevent some forms of apoptosis from ensuing, Polkand co-workers showed that inactivation of KSR in YAMC colon cellsantagonizes the anti-apoptotic signals emanating from TNF receptor,converting TNF into an inducer of apoptosis in these cells (41,42).

In the present example, we confine our studies to inactivation of theEGFR/Ras pathway as a proof-in-principle that this strategy can lead toradiosensitization. To explore this venue, we synthesized KSR specificAS-ODNs to pharmacologically inhibit KSR function (as described hereinabove) and employed A431 cells, which are driven through wild type Rasby an 100-fold overexpression of activated EGFR. When tested in vitro,AS-ODNs were specifically taken up into the nucleus, resulting indecreased endogenous KSR gene expression and inhibition of A431proliferation, invasion, transformation and tumorigenesis (see Example2). Moreover, here we demonstrate that inactivation of KSR via genetic(expression of KSR-AS) or pharmacological (AS-ODNs) approaches yieldssensitization of A431 cells to IR-induced apoptosis in vitro.

Results

AS-KSR and DN Ki-KSR sensitize A431 cells to IR-induced apoptosis: Totest the hypothesis that inactivation of KSR might enhance thesensitivity to IR-induced cell killing, A431 cells stably-transfectedwith different KSR constructs were analyzed for their sensitivity toIR-induced apoptosis. A431 cells, due to the overexpression of activatedEGFR, are known to be radioresistant (43). Serum-starved A431 cells wereirradiated with a single dose of 20 Gy and apoptosis was deter-mined 24hr post-IR by flow cytometry using Annexin V-FITC (Sigma). FIG. 19 showsthat A431 cells overexpressing wild-type Flag-KSR (KSR-S) displayedminimal IR-induced apoptosis. Expression of Flag-AS-KSR (KSR-AS) ordominant-negative Flag-Ki-KSR (Ki-KSR) radiosensitized A431 cells,resulting in a substantial increase in IR-induced apoptosis (n=3).Nearly 40% of A431-KSR-AS and Ki-KSR cells underwent apoptosis 24 hrafter IR. These results, although preliminary, strongly suggest that KSRmay play a key role in cellular sensitivity to IR. Comparable resultswere obtained when apoptosis was scored with Bisbenzimide staining.

KSR AS-ODNs Sensitize A431 Cells to IR-induced Apoptosis:

As described above, abrogation of KSR function by a genetic approach(AS-KSR and DN-Ki-KSR) sensitized A431 cells to IR-induced apoptosis. Totest the effectiveness of pharmacologic inhibition of KSR1 geneexpression by AS-ODN on radiosensitivity, A431 cells were treated with200 nM of AS-214231 for 36 hr prior to IR. Apoptosis was quantitated byflow-cytometry using Annexin V-FITC. As shown in FIG. 20, AS-214231sensitized A431 cells, leading to a 2-fold increase in IR-inducedapoptosis after 72 hours. The magnitude of this effect is comparable tothat observed with overexpression of AS-KSR. In contrast, control ODNhad no significant effect on radiation sensitivity to IR-inducedapoptosis. It should be noted that at the concentration ofOligofectamine used, no significant elevation of basal level ofapoptosis was detected (not shown). These results indicate that theradiosensitizing effect of AS-214231 is KSR sequence specific and thatit might be feasible to use KSR AS-ODNs as radiosensitizers.

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This invention may be embodied in other forms or carried out in otherways without departing from the spirit or essential characteristicsthereof. The present disclosure is therefore to be considered as in allaspects illustrate and not restrictive, the scope of the invention beingindicated by the appended Claims, and all changes which come within themeaning and range of equivalency are intended to be embraced therein.

Various references are cited throughout this Specification, each ofwhich is incorporated herein by reference in its entirety.

1. An oligonucleotide which is substantially complementary to a regionof KSR RNA, wherein said oligonucleotide inhibits the expression of KSR.2. The oligonucleotide of claim 1 which is substantially complementaryto a nucleic acid encoding mammalian KSR.
 3. The oligonucleotide ofclaim 1 which is substantially complementary to a nucleic acid encodinghuman KSR.
 4. An oligonucleotide which is substantially complementary toa translation initiation site, 5′ untranslated region, coding region or3′ untranslated region of mRNA encoding mammalian KSR.
 5. An antisenseoligonucleotide comprising a sequence substantially complementary to theCA1 region of KSR.
 6. An antisense oligonucleotide comprising a sequencesubstantially complementary to nucleotides 124 to 243 (SEQ ID NO: 1) ofthe coding sequence of mouse KSR or nucleotides 97 to 216 of human KSR(SEQ ID NO: 25), or a portion thereof.
 7. The oligonucleotide of claim 6comprising a sequence substantially complementary to nucleotidesselected from the group of: (a) 124 to 141 of the sequence of human KSR,corresponding to 151 to 168 of the sequence of mouse KSR (SEQ ID NO:3),(b) 154 to 171 of the sequence of human KSR (SEQ ID NO:27) (c) 181 to198 of the sequence of mouse KSR (SEQ ID NO: 4); and (d) 187 to 204 ofthe sequence of human KSR, corresponding to 214 to 231 of the sequenceof mouse KSR (SEQ ID NO: 5).
 8. An antisense oligonucleotide comprisinga sequence selected from the group of SEQ ID NOS: 6-8 and SEQ ID NOS:29-38.
 9. The oligonucleotide of claim 1 labeled with a detectablelabel.
 10. The oligonucleotide of claim 1 wherein the label is selectedfrom enzymes, ligands, chemicals which fluoresce and radioactiveelements.
 11. The oligonucleotide of claim 1 wherein saidoligonucleotide comprises at least one phosphorothioate linkage.
 12. Arecombinant DNA molecule comprising a nucleic acid sequence whichencodes on transcription an antisense RNA complementary to mammalian KSRRNA or a portion thereof.
 13. The recombinant DNA molecule of claim 12wherein said nucleic acid sequence is operatively linked to atranscription control sequence.
 14. A cell line transfected with therecombinant DNA molecule of claim
 13. 15. An expression vector capableof expressing a nucleic acid which is substantially complementary to thecoding sequence of KSR RNA, or a portion/fragment thereof, wherein saidoligonucleotide/nucleic acid inhibits the expression of KSR.
 16. Anexpression vector capable of expressing an oligonucleotide which issubstantially complementary to the CA1 region of the coding sequence ofKSR RNA, or a portion/fragment thereof, wherein said oligonucleotideinhibits the expression of KSR.
 17. A pharmaceutical compositioncomprising a therapeutically effective amount of an antisenseoligonucleotide of claim 1 and a pharmaceutically acceptable carrier ordiluent.
 18. A composition comprising the oligonucleotide of claim 1 anda pharmaceutically acceptable carrier or diluent.
 19. A compositioncomprising one or more chemotherapeutic or radiotherapeutic agent and anoligonucleotide which is targeted to a mRNA encoding mammalian KSR andwhich inhibits KSR expression.
 20. A composition comprising anexpression vector and a pharmaceutically acceptable carrier or diluent,wherein said expression vector is capable of expressing nucleic acidwhich is substantially complementary to the coding sequence of KSR RNA,or a portion/fragment thereof, wherein said nucleic acid inhibits theexpression of KSR.
 21. A method of inhibiting the expression ofmammalian KSR comprising contacting cells which express KSR with aneffective amount of a nucleic acid which is complementary to a portionof the mRNA encoding KSR.
 22. A method of inhibiting the expression ofmammalian KSR comprising contacting cells which express KSR with aneffective amount of the oligonucleotide of claim 1 whereby expression ofmammalian KSR is inhibited.
 23. A method of treating or preventing ahypeiproliferative condition associated with the expression of gƒ-Ras orheightened expression of Ras in a mammal comprising administering tosaid mammal a therapeutically effective amount of a compound or agentwhich inhibits the expression of mammalian KSR protein.
 24. The methodof claim 23 wherein said compound or agent is an antisenseoligonucleotide which specifically hybridizes to a portion of the mRNAencoding KSR.
 25. A method of treating or preventing ahyperproliferative condition associated with the expression of gƒ-Ras orheightened expression of Ras in a mammal comprising expressing in saidmammal or administering to said mammal a therapeutically effectiveamount of a nucleic acid which is complementary to a portion of the mRNAencoding KSR.
 26. A method of treating or inhibiting the progression ofcancer in a mammal comprising administering to a mammal atherapeutically effective amount of a compound or agent which inhibitsthe expression of mammalian KSR protein.
 27. The method of claim 26,wherein said cancer is selected from the group of pancreatic cancer,lung cancer, skin cancer, urinary tract cancer, bladder cancer, livercancer, thyroid cancer, colon cancer, intestinal cancer, leukemia,lymphoma, neuroblastoma, head and neck cancer, breast cancer, ovariancancer, stomach cancer, esophageal cancer and prostate cancer.
 28. Amethod of treating or inhibiting the progression of cancer in a mammalcomprising administering to a mammal a therapeutically effective amountof the oligonucleotide of claim
 1. 29. A method of conferringradiosensitivity to ionizing radiation in tumor cells in a mammalcomprising administering to a mammal a therapeutically effective amountof a compound or agent which inhibits the expression of mammalian KSRprotein.
 30. A method of conferring radiosensitivity to ionizingradiation in tumor cells in a mammal comprising administering to amammal a therapeutically effective amount of the oligonucleotide ofclaim
 1. 31. The method of claim 29 or 30 wherein the tumor cells arecancer cells selected from the group of pancreatic cancer, lung cancer,skin cancer, urinary tract cancer, bladder cancer, liver cancer, thyroidcancer, colon cancer, intestinal cancer, leukemia, lymphoma,neuroblastoma, head and neck cancer, breast cancer, ovarian cancer,stomach cancer, esophageal cancer and prostate cancer.
 32. A method ofidentifying compounds or agents which inhibit the expression of KSRcomprising the steps of: (c) incubating a cell expressing KSR in thepresence and absence of a candidate compound or agent; and (d) detectingor measuring the expression of KSR in the presence and absence of acandidate compound or agent, whereby a decrease in the expression of KSRin the presence of said candidate compound or agent versus in theabsence of said candidate compound or agent indicates that said compoundor agent inhibits the expression of KSR.
 33. A ribozyme that cleaves KSRmRNA.